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Prion 2015 Oral Abstracts

Propagation

O.01: Transgenic mice expressinghuman wild-type a-synuclein developneuropathology after inoculation withbrain homogenates from patients with

multiple system atrophy or aged subjectswithout neurological disorder

Maria Eugenia Bernis1, Julius TachuBabila1, Sara Breid1, Ullrich W€ullner1,2,

and G€ultekin Tamg€uney1

1German Center for Neurodegenerative Diseases

(DZNE); Bonn, Germany; 2Department of

Neurology; University of Bonn; Bonn, Germany

Multiple system atrophy (MSA) andParkinson’s disease are synucleinopathies thatare defined by the presence of aggregated andhyperphosphorylated a-synuclein (a-syn) withincells of the central nervous system (CNS).

Recent findings suggest that pathologicala-syn may spread prion-like within the nervoussystem. We investigated prion-like propagationof pathological a-syn in Tg(SNCA)1Nbm/Jmice that do not express mouse but low levelsof human wt a-syn and do not naturallydevelop any pathology or neurodegenerativedisease.

We inoculated brain homogenate from2 patients with MSA, from 2 aged control sub-jects without neurological disorder, or salineintrastriatally into Tg(SNCA)1Nbm/J mice.Challenged mice were sacrificed at 90, 180,and 270 d post inoculation and were analyzedbiochemically and immunohistochemically forpathological a-syn.

Brain homogenates from MSA or agedcontrol subjects but not saline triggered

progressive accumulation of aggregated a-synin neurons of inoculated mice. Aggregates ofa-syn were hyperphosphorylated and co-stained for p62 that targets proteins for degra-dation. Aggregates of pathological a-syn werefirst observable in the ipsilateral brain hemi-sphere and over time in the contralateral hemi-sphere and in more rostral and caudal areas.

Our findings show that brain homogenatefrom MSA patients but not saline induces path-ological changes in the CNS of Tg(SNCA)1Nbm/J mice. Our data support that pathologi-cal a-syn may propagate prion-like along neu-ronal networks. Furthermore, human wt a-synsupports propagation of pathological a-syn.Intriguingly, brain homogenate from aged con-trol subjects without neurological disorderequally induced synucleinopathy in brains ofTg(SNCA)1Nbm/J mice suggesting that agedhuman brains can contain pathological a-syn.

O.02: Experimental transmissibility ofmutant SOD1 motor neuron disease

Jacob Ayers, Susan Fromholt, MorganKoch, Adam DeBosier, Ben McMahon,

Guilian Xu, and David Borchelt

University of Florida; Gainesville, FL USA

By unknown mechanisms, the symptoms ofamyotrophic lateral sclerosis (ALS) seem tospread along anatomical pathways to engulf themotor nervous system. The rate at which symp-toms spread is one of the primary drivers of dis-ease progression. One mechanism by whichALS symptoms could spread is by a prion-likepropagation of a toxic misfolded protein fromcell to cell along anatomic pathways. Proteinsthat can transmit toxic conformations between

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Prion, 9:S1–S10, 2015ISSN: 1933-6896 print / 1933-690X online

cells often can also experimentally transmit dis-ease between individual organisms. To surveythe ease with which motor neuron disease(MND) can be transmitted, we injected spinalcord homogenates prepared from paralyzedmice expressing mutant superoxide dismutase 1(SOD1-G93A and G37R) into the spinal cordsof genetically vulnerable SOD1 transgenicmice. From the various models we tested, oneemerged as showing high vulnerability. Tissuehomogenates from paralyzed G93A expressingmice induced MND in 6 of 10 mice expressinglow levels of G85R-SOD1 fused to yellow fluo-rescent protein (G85R-YFP mice) by 3–11 months, and produced widespread spinalinclusion pathology. Importantly, second pas-sage of homogenates from G93A!G85R-YFPmice back into newborn G85R-YFP mice,induced disease in 4 of 4 mice by 3 months ofage. Homogenates from paralyzed miceexpressing the G37R variant were among thosethat transmitted poorly regardless of the strainof recipient transgenic animal injected, a findingsuggestive of strain-like properties that manifestas differing abilities to transmit MND. Together,our data provide a working model for MNDtransmission to study the pathogenesis of ALS.

O.03: Role of brain interstitial fluid flowin very early generation and spread ofPrPres after microinjection of prioninfectivity in C57BL mouse brain

Bruce Chesebro, James Striebel, AlejandraRangel, Katie Phillips, Andrew Hughson,

Byron Caughey, and Brent Race

Rocky Mountain Laboratory; Hamilton, MT USA

Prion infectivity typically spreads alongneurons following the paths of neuron cir-cuitry within the CNS; however, less is knownabout the process of initial brain infection.Here we studied spread of disease-associatedPrPres in brain of C57BL mice from30 minutes to 40 d after microinjection(0.5 ml) of scrapie infectivity into the stria-tum. After microinjection of 22L scrapie,PrPres was visible in the needle track and

around nearby blood vessels at 30 min in bothC57BL and Prnp-null (KO) control mice. Thisrapid initial spread to vessels appeared to bevia brain interstitial fluid (ISF) flow in peri-vascular and periaxonal regions. At 3 dpi inKO mice, inoculated PrPres was mostly gone.Remarkably, at 3 and 7 dpi in C57BL mice,generation of new PrPres was detectable byimmunohistochemistry, immunoblot and RT-QUIC assay. Again, PrPres was associatedwith perivascular sites of ISF drainage, andPrPres associated mainly with perivascularastroglia, and only minimally with neurons,microglia and oligodendroglia. By 20–40 dpi,PrPres had spread to ipsilateral thalamus andcerebral cortex at locations 1.5–2.5 mm dis-tant from the injection site. Both the ipsilat-eral PrPres distribution and the rapid transittime suggested that spread from striatum wasvia neuronal circuity. Thus early 22L scrapiespread involved both neurons and ISF flow,however PrPres mainly accumulated in astro-glia. In experiments using scrapie strain ME7,similar spreading mechanisms were observed,but PrPres accumulated primarily in associa-tion with neuronal cell bodies and neuropil,which was not seen with strain 22L.

O.04: A single coding polymorphismin the PRNP gene significantly alters

the transmission dynamicsof blood-borne prions

Fiona Houston, Richard Alejo Blanco,Sandra McCutcheon, Boon Chin Tan,

Allister Smith, and Jean Manson

University of Edinburgh; Edinburgh, UK

Sheep experimentally infected with bovinespongiform encephalopathy (BSE) have pro-vided a useful model in which to study the risksof transmission of prion disease by transfusionof blood components. We have previouslyshown that several components relevant tohuman clinical practice (red cell concentrate,platelets, plasma) are infectious, and that leuco-depletion of these components does notcompletely prevent disease transmission.

S2 Prion 2015 Oral Abstracts

Further analysis of the data has identified factorswhich distinguish donor sheep that transmittedinfection via blood components (“transmitters”),from those that did not (“non-transmitters”).The codon 141 polymorphism of the sheepPRNP gene (L ! F) was strongly associatedwith the probability of transmitting infection,with the majority of transmitters having a141LL genotype, while the majority of non-transmitters were 141LF. Since codon141 genotype has also been associated with var-iation in incubation period, this finding mayreflect the stage of the incubation period reachedby the donor at the time when blood was col-lected, because titres of infectivity in blood tendto increase as animals progress toward the clini-cal phase of infection. Another factor thatappears to differ between transmitting and non-transmitting donors is the extent of PrPd deposi-tion in lymphoid tissues, with transmitters hav-ing a higher proportion of positive lymphoidtissues than non-transmitters, regardless of theircodon 141 genotype. This suggests that a singlenucleotide polymorphism of PRNP, and/or theextent of prion replication in lymphoid tissues,may influence titres of blood-borne infectivityand thus the risk of disease transmission bytransfusion of blood components.

O.05: Transmission of prions to primatesafter extended silent incubation periods:Implications for BSE and scrapie riskassessment in human populations

Emmanuel Comoy, Jacqueline Mikol,Val�erie Durand, Sophie Luccantoni,Evelyne Correia, Nathalie Lescoutra,

Capucine Dehen, and Jean-Philippe Deslys

Atomic Energy Commission; Fontenay-aux-Roses,

France

Prion diseases (PD) are the unique neurode-generative proteinopathies reputed to be trans-missible under field conditions since decades.The transmission of Bovine SpongiformEncephalopathy (BSE) to humans evidencedthat an animal PD might be zoonotic under

appropriate conditions. Contrarily, in theabsence of obvious (epidemiological or experi-mental) elements supporting a transmission orgenetic predispositions, PD, like the other pro-teinopathies, are reputed to occur spontane-ously (atpical animal prion strains, sporadicCJD summing 80% of human prion cases).

Non-human primate models provided thefirst evidences supporting the transmissibiity ofhuman prion strains and the zoonotic potentialof BSE. Among them, cynomolgus macaquesbrought major information for BSE risk assess-ment for human health (Chen, 2014), accordingto their phylogenetic proximity to humans andextended lifetime. We used this model to assessthe zoonotic potential of other animal PD frombovine, ovine and cervid origins even aftervery long silent incubation periods.

We recently observed the direct transmissionof a natural classical scrapie isolate to macaqueafter a 10-year silent incubation period, withfeatures similar to some reported for humancases of sporadic CJD, albeit requiring fourfoldlonge incubation than BSE. Scrapie, as recentlyevoked in humanized mice (Cassard, 2014), isthe third potentially zoonotic PD (with BSEand L-type BSE), thus questioning the origin ofhuman sporadic cases. We will present anupdated panorama of our different transmissionstudies and discuss the implications of suchextended incubation periods on risk assessmentof animal PD for human health.

O.06: Prion properties of theAlzheimer’s disease associated proteins

in the yeast model

Yury Chernoff1,2, PavithraChandramowlishwaran1, Kristin Casey1,Abigail Harrover1, Daniil Kachkin2,

Alexandr Rubel2, and Alexey Galkin2,3

1Georgia Institute of Technology; Atlanta, GA USA;2St. Petersburg State University; St. Petersburg,

Russia; 3St. Petersburg Branch of RAS Institute of

General Genetics; St. Petersburg, Russia

Amyloid formation is implicated in varioushuman diseases, and many amyloids are

Prion 2015 Oral Abstracts S3

suspected to possess transmissible (prion) prop-erties. However, molecular mechanisms ofamyloid formation and propagation are difficultto investigate in vivo due to complexity of thehuman organism. We have established a yeastmodel for studying the prion properties ofmammalian (including human) proteins. Ourmodel employs chimeric constructs, containingthe mammalian amyloidogenic proteins (ordomains) fused to various fragments of theyeast prion protein Sup35. Phenotypic and bio-chemical detection assays, previously devel-oped for the Sup35 prion, enable us to detectprion nucleation and propagation by mamma-lian proteins. By using this approach, we haveinvestigated prion properties of Abeta and tauproteins. Oligomerization and aggregation ofAbeta and tau is known to be associated withAlzheimer’s disease in humans. We haveshown that both proteins confer prion charac-teristics to the chimeric constructs in yeast.Effects of known pro-aggregation and anti-aggregation mutations in these proteins onprion formation in yeast generally correspondto their effects on the disease development inhumans. For example, the D23N substitution inAbeta increases prion nucleation in the yeastsystem. New mutations with predicted effectson amyloidogenesis are being generated andtested. Formation of different prion “strains”by chimeric proteins has been detected in yeast.Assays for studying interactions between dif-ferent amyloidogenic proteins in the yeast cellhave been developed. Overall, yeast model ena-bles us to perform genetic dissection of molec-ular processes leading to the initiation ofAlzheimer’s disease.

Topics in Animal Prions

O.07: 2-aminothiazole treatment ofchronic wasting disease in transgenic

mice expressing elk PrP

David Berry, Kurt Giles, Abby Oehler,Sumita Bhardwaj, Stephen DeArmond, and

Stanley Prusiner

University of California, San Francisco, CA USA

Treatment with the 2-aminothiazole IND24extended the survival of wild-type miceinfected with mouse-passaged RML scrapieprions, but also resulted in the emergence of adrug-resistant prion strain. IND24 treatmentwas also efficacious against Tg mice expressingElkPrP and infected with chronic wasting dis-ease (CWD) prions, but the impact of treatmenton the properties of CWD prions was not deter-mined. Here, we assessed whether IND24 treat-ment extended the survival of additionalnatural isolates in Tg mice infected with sheepscrapie or CWD prions using 2 isolates foreach disease. Multiple IND24 treatment regi-mens doubled the incubation times for CWD-infected mice, but IND24 treatment had noeffect on the survival of the ovine scrapie-infected mice. Biochemical, neuropathologic,and cell culture analyses were used to charac-terize the prion strain properties followingtreatment, and indicated that the CWD prionswere not altered by IND24 treatment regardlessof survival extension. A second passage in theabsence of treatment reproduced the originalsurvival time, and cells infected with prionsfrom animals that were treated with IND24were as susceptible to IND24 treatment asCWD prions that were never before exposed toIND24. These results suggest that IND24 maybe a viable candidate for treating CWD ininfected captive cervid populations, and raisequestions about why some strains acquire resis-tance upon treatment whereas others do not.


O.08: H-type bovine spongiformencephalopathy associated with E211Kprion protein polymorphism: Clinical

and pathologic features in wild-type andE211K cattle following intracranial

inoculation

S Jo Moore, M Heather West Greenlee,Jodi Smith, Eric Nicholson, Cathy Vrentas,

and Justin Greenlee

United States Department of Agriculture; Ames,

IA USA

In 2006 an H-type bovine spongiformencephalopathy (BSE) case was reported in ananimal with an unusual polymorphism(E211K) in the prion protein gene. Althoughthe prevalence of this polymorphism is low,cattle carrying the K211 allele are predisposedto rapid onset of H-type BSE when exposed.The purpose of this study was to investigate thephenotype of this BSE strain in wild-type(E211E) and E211K heterozygous cattle.

One calf carrying the wild-type allele andone E211K calf were inoculated intracraniallywith H-type BSE brain homogenate from theUS 2006 case that also carried one K211 allelle.In addition, one wild-type calf and one E211Kcalf were inoculated intracranially with brainhomogenate from a US 2003 classical BSEcase. All animals succumbed to clinical dis-ease. Survival times for E211K H-type BSEinoculated catttle (10 and 18 months) wereshorter than the classical BSE inoculated cattle(both 26 months). Significant changes in retinalfunction were observed in H-type BSE chal-lenged cattle only. Animals challenged with thesame inoculum showed similar severity andneuroanatomical distribution of vacuolationand disease-associated prion protein depositionin the brain, though differences in neuropathol-ogy were observed between E211K H-typeBSE and classical BSE inoculated animals.Western blot results for brain tissue from chal-lenged animals were consistent with the inocu-lum strains.

This study demonstrates that the phenotypeof E211K H-type BSE remains stable when

transmitted to cattle without the E211K poly-morphism, and exhibits a number of featuresthat differ from classical BSE in both wild-typeand E211K cattle.

Mechanisms of Disease

O.09: Mutant prion proteins related togenetic prion diseases impair

intracellular trafficking

Elena Restelli1, Vanessa Capone2,Manuela Pozzoli1, Davide Ortolan1,

Michele Sallese2, and Roberto Chiesa1

1Department of Neuroscience; IRCCS - Mario

Negri Institute for Pharmacological Research;

Milano, Italy; 2Unit of Genomic Approaches to

Membrane Traffic; Mario Negri Sud Foundation;

Santa Maria Imbaro (CH), Italy

Fatal familial insomnia (FFI), geneticCreutzfeldt-Jakob disease (gCJD) and Gerst-mann-Str€aussler-Scheinker (GSS) syndromeare neurodegenerative disorders linked to muta-tions in the prion protein (PrP) gene. The mech-anism of neurotoxicity of mutant PrP is notclear, but misfolding and intracellular accumu-lation may contribute to the pathogenic process.We previously found that mouse (mo) PrPD177N/M128, homologous to the D178N/M129 mutation associated with FFI, accumu-lates in the Golgi of neuronal cells, impairingpost-Golgi trafficking.1 To further characterizethe trafficking defect induced by the FFI muta-tion, and test whether other mutants had similareffects, we analyzed the efficiency of secretorytransport in cells expressing moPrP D177N/M128, D177N/V128 (homologous to theD178N/V129 mutation linked to gCJD), andPG14, a 9-octapeptide repeat insertion associ-ated with GSS. We used transfected HeLa cells,embryonic fibroblasts and primary neuronsfrom transgenic mice, as well as human fibro-blasts from carriers of the FFI mutation. In allcell models mutant PrPs showed abnormalintracellular localizations, accumulating in dif-ferent compartments of the secretory pathway.To test the efficiency of the membrane


trafficking system, we monitored the intracellu-lar transport of the temperature-sensitive vesic-ular stomatite virus glycoprotein (VSVG), awell-established cargo reporter. We observedmarked alterations in secretory transport, withVSVG accumulating mainly in the Golgi com-plex. Our results indicate that different patho-genic mutations share the property of impairingintracellular trafficking. This suggests thatdefective intracellular transport may be a gen-eral mechanism of neurotoxicity in geneticprion diseases.

1. Massignan T, Biasini E, Lauranzano E, Veglianese

P, Pignataro M, Fioriti L, Harris DA, Salmona M,

Chiesa R, Bonetto V. Mutant prion protein expression

is associated with an alteration of the Rab GDP

dissociation inhibitor alpha (GDI)/Rab11 pathway.

Mol Cell Proteomics 2010; 9(4):611–622; PMID:

19996123; http://dx.doi.org/10.1074/mcp.M900271-

MCP200

O.10: The sheddase ADAM10significantly impacts on prion disease

Hermann Altmeppen1, Johannes Prox2,Susanne Krasemann1, Berta Puig1,

Katharina Kruszewski1, Frank Dohler1,Christian Bernreuther1, Ana Hoxha1,Luise Linsenmeier1, Beata Sikorska3,

Pawel Liberski3, Udo Bartsch1,Paul Saftig2, and Markus Glatzel1

1University Medical Center Hamburg-Eppendorf;

Hamburg, Germany; 2Christian-Albrechts

University; Kiel, Germany; 3Medical University;

Lodz, Poland

Proteolytic processing of key proteins, be itdeleterious or protective, is relevant in manyneurodegenerative proteinopathies such asAlzheimer’s or prion disease. With regard tothe latter, while a-cleavage in the middle of thecellular prion protein (PrPC) has been shown toimpair misfolding into the pathogenic isoform(PrPSc) and thus to be protective against priondisease, the role of another physiological cleav-age (i.e., shedding in close proximity to theGPI-anchor of PrPC) remained largelyunknown. We and others have identified

ADAM10 as the physiologically relevant shed-dase of PrPC regulating its membranehomeostasis.

Using a novel mouse model, we show thatdepletion of ADAM10 in forebrain neuronsleads to posttranslational increase of PrPC lev-els. When infected with prions, these micepresent with drastically shortened incubationtimes, increased PrPSc formation and upregula-tion of calpain. Our spatiotemporal analysesalso suggest that absence of shedding impairsspread of prion pathology within the brain.Moreover, our mouse model provides someinteresting insights into central issues discussedin the prion field, such as (i) an inhibitory effectof anchorless PrP versions on the conversionprocess, (ii) mechanisms of prion-associatedneurotoxicity, and (iii) a likely disparitybetween PrPSc amounts and prion infectivity.

Taken together, ADAM10-mediated shed-ding seems to have a dual role in prion diseasesthus emphasizing the relevance of proteolyticprocessing in these conditions. Given the sug-gested role of PrPC as a receptor for toxic pro-tein oligomers in more commonproteinopathies our findings might impact onthese devastating conditions as well.

Structure/Function

O.11: Structural determinants ofphenotypic diversity and replication rate

of human prions

Jiri Safar, Xiangzhu Xiao,Mohammad Kabir, Shugui Chen,Chae Kim, Tracy Haldiman,

Yvonne Cohen, Wei Chen, Mark Cohen,and Witold Surewicz

Case Western Reserve University; Cleveland,

OH USA

The infectious pathogen responsible forprion diseases is the misfolded, aggregatedform of the prion protein, PrPSc. In contrast torecent progress in studies of laboratory rodent-adapted prions, current understanding of the


molecular basis of human prion diseases and,especially, their vast phenotypic diversity isvery limited. Here, we have purified proteinaseresistant PrPSc aggregates from two major phe-notypes of sporadic Creutzfeldt-Jakob disease(sCJD), determined their conformational stabil-ity and replication tempo in vitro, as well ascharacterized structural organization usingrecently emerged approaches based on hydro-gen/deuterium (H/D) exchange coupled withmass spectrometry. Our data clearly demon-strate that these phenotypically distant prionsdiffer in a major way with regard to their struc-tural organization, both at the level of the poly-peptide backbone (as indicated by backboneamide H/D exchange data) as well as the qua-ternary packing arrangements (as indicated byH/D exchange kinetics for histidine sidechains). Furthermore, these data indicate that,in contrast to previous observations on yeastand some murine prion strains, the replicationrate of sCJD prions is primarily determined notby conformational stability but by specificstructural features that control the growth rateof prion protein aggregates.

O.12: Scrapie-specific C-terminalantibody reveals conformationaldifferences between prion strains

Eri Saijo1, Andrew Hughson1,Gregory Raymond1, Motohiro Horiuchi2,

and Byron Caughey1

1Laboratory of Persistent Viral Diseases; Rocky

Mountain Laboratories; National Institutes of

Allergy and Infectious Diseases; National Institutes

of Health; Hamilton, MT USA; 2Laboratory of

Veterinary Hygiene; Graduate School of Veterinary

Medicine; Hokkaido University; Sapporo, Japan

Misfolding of the cellular form of prionprotein (PrPC) into the pathological PrP(PrPSc) in the brain can lead to transmissiblespongiform encephalopathies or prion dis-eases in humans and animals. PrPC is analpha-helix-rich monomer, while PrPSc formsbeta-sheet-rich oligomers and amyloid fibrils.Prion strains have been classified based on

different incubation times, neuropathologicallesions and biochemical characteristics.However, the detailed structure of PrPSc andconformational differences between strainsare poorly understood. In this study, we usedantibodies to different epitopes to probe thestructures of PrPSc isolated from brains ofmice with either the Chandler or 22L strainsor hamsters with 263K scrapie. Epitope map-ping of PrPSc was performed under native orguanidine-denatured conditions by indirect-ELISA. Our results showed that only a smallsubset of antibodies recognized epitopes inthe native structure of PrPSc. One of thoseantibodies, with a conformationally sensitiveC-terminal epitope, had strongly differingreactivities to the native Chandler and 22Lstrains of PrPSc despite the fact that thesemurine prions share the same PrP primarystructure. Although it has long been apparentthat prion strains can differ conformationallynear the N-termini of molecules forming theproteinase-K resistant core of PrPSc, ourresults show evidence for stain-dependentconformational differences near the C-termi-nal as well.

O.13: Misfolded wild-type SOD1 inducedby pathological FUS or TDP-43transmits intercellularly and is

propagation-competent

Edward Pokrishevsky, Leslie Grad, andNeil Cashman

University of British Columbia; Vancouver,

BC, Canada

Clinically indistinguishable cases of amyo-trophic lateral sclerosis (ALS) can be causedby either inheritable mutation in the genesencoding SOD1, TDP-43, FUS, among others,or can occur sporadically. Misfolded SOD1 hasbeen detected in both familial and sporadicALS patients, despite SOD1 mutationsaccounting for only »2% of total cases. Wepreviously reported that pathological FUS orTDP-43 kindles misfolding of human wild-type(wt) SOD1 in living cells. Here, we use human


cell cultures and mouse primary neural culturesexpressing human wtSOD1, to establish thatFUS or TDP-43-induced misfolded SOD1 cantraverse between cells through the incubationof untransfected cells with conditioned media,triggering conversion of endogenous SOD1 ina prion-like fashion. This intercellular spread isarrested by pre-incubation of the conditionedmedia with misfolded SOD1-specific antibod-ies, demonstrating their therapeutic potential.We find that recipient cells pre-treated withSOD1-siRNA do not contain misfolded SOD1,indicating that endogenous SOD1 is required assubstrate for active conversion. Our data alsoshows that conditioned media obtained frommutant TDP-43 and FUS transfected, or wild-type TDP-43 over-expressing, cells is cytotoxicto the recipient cells. Furthermore, transfectionof TDP-43 into cells triggers its cleavage, mis-localization and hyperphosphorylation; theseproperties are not observed in untransfectedcells incubated with conditioned media frompathological TDP-43 transfected cells, furtherconfirming that the transmission of SOD1 mis-folding occurs independently of TDP-43.

O.14: The architecture of recombinantprions is similar to that of brain-derivedprions: Insights from limited proteolysis

Alejandro M Sevillano1,Natalia Fern�andez-Borges2,

Neelam Younas1,3,Ester V�azquez-Fern�andez4,

Saioa R Elezgarai2, Hasier Era~na2,Romolo Nonno5, Joaqu�ın Castilla2,6, and

Jes�us R Requena1

1CIMUS Biomedical Research Institute; University

of Santiago de Compostela-IDIS; Santiago de

Compostela, Spain; 2CIC bioGUNE; Derio, Spain;3Prion Research Group; Dept. of Neurology;

Universit€ats Medizin G€ottingen; Gottingen,Germany; 4Centre for Prions and Protein Folding

Diseases; University of Alberta; Edmonton,

Canada; 5Department of Veterinary Public Health

and Food Safety; Istituto Superiore di Sanit�a;Rome, Italy; 6IKERBASQUE; Basque Foundation

for Science; Vitoria, Spain

Substantial evidence suggests that PrPSc is a4-rung b-solenoid, and that individual PrPSc

subunits stack to form amyloid fibers. Werecently used limited proteolysis to map theb-strands and connecting loops that conformthe PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope mapping, and massspectrometry, we identified positions »117/119, 133–134, 152–153, 141, 162, 169 and 179as PK cleavage sites in PrPSc. Such sites defineloops and/or borders of b strands, and are help-ing us define the threading of the b-solenoid.

We have now extended this approach torecombinant PrPSc (recPrPSc). We apply theterm recPrPSc to bona fide recombinant prionsprepared by PMCA, exhibiting infectious prop-erties with attack rates of 100%.

Limited proteolysis of a variety of mouseand bank vole recPrPSc species, prepared underslightly different conditions, yields the same N-terminally truncated PK-resistant fragmentsseen in brain-derived PrPSc, indicative of anoverall similar architecture of both prion types.However, lower resistance to PK and a compar-atively higher abundance of smaller fragmentswith respect to the “canonic” »90–230 PK-resistant core, suggests higher flexibility andnuances in threading for recPrPSc. Furthermore,doubly N- and C-terminally truncated frag-ments, in particular »90–152, are oftendetected; similar fragments are characteristic ofatypical strains of brain-derived PrPSc.

Ongoing comparison of specific digestionpatterns (relative abundances of individualfragments) and incubation times will allowextracting conclusions on relationshipsbetween structure and biological properties ofdifferent recPrPSc species. Recombinant PrPSc

offers exciting opportunities for structural stud-ies not possible to date with brain-derivedPrPSc.


O.15: In a mammalian model ofepithelial-to-mesenchymal transition the

prion protein mediates b-catenin-dependent transcriptional activation of a

key EMT regulator

MMehrabian1,2, D Brethour1,2

H Wang,1 Z Xi,1 E Rogaeva1,3, andG Schmitt-Ulms1,2

1Tanz Centre for Research in Neurodegenerative

Diseases; University of Toronto; Toronto, Ontario,

Canada; ; 2Department of Laboratory Medicine

University of Toronto; Toronto, Ontario, Canada;

3Department of Medicine, University of Toronto,

Ontario, Canada

The physiological function of the prion pro-tein (PrP) has remained elusive despite itswidely recognized role in neurodegenerativediseases and sustained efforts to understand itsmolecular biology. On the basis of its evolu-tionary relationship to ZIP zinc transportersand the characteristics of a gastrulation arrestphenotype in a PrP-deficient zebrafish model,we considered that PrP may contribute to themorphogenetic reprogramming of cells under-lying epithelial-to-mesenchymal (EMT) transi-tions. We now report that consistent with thishypothesis, PrP levels can be observed toincrease more than 5-fold during EMT, and itsCRISPR-Cas9-mediated knockout interfereswith EMT in NMuMG cells, a widely usedmouse model for studying this cellular pro-gram. Subsequent endeavors to dissect themolecular underpinnings of this phenotyperevealed that PrP-deficient cells fail to executean essential step during EMT. Surprisingly, thisimpairment was caused by a failure of PrP-defi-cient cells to activate transcription of a criticalEMT mediator. A subsequent comparativeglobal proteome analysis of wild-type and PrP-deficient NMuMG cells pointed toward b-cate-nin as a transcriptional regulator that contrib-utes to this deficiency of PrP-knockout cells.Indeed, pharmacological blockade or siRNA-based knockdown of b-catenin mimicked PrP-deficiency. By placing PrP in a signaling path-way that is essential for EMT in NMuMG cells,

our study introduces an easily accessible modelfor studying signaling downstream of PrP, andprovides a fresh angle for understanding therole of PrP in health and disease.

O.16: Effect of cellular prion onneurogenesis after acute injury and

chronic prion infection

Lindsay Parrie, Jenna Crowell, andRichard Bessen

Colorado State University; Fort Collins, CO USA

The cellular prion protein (PrPC) has beenassociated with varied biological processesincluding cell signaling and neuroprotection,yet its physiological function(s) remain ambig-uous. The goal of this study is to determine therole of PrPC in adult neurogenesis using themurine olfactory system model. Olfactory sen-sory neurons (OSNs) within the olfactory sen-sory epithelium (OSE) undergo continualneurogenesis, integration, and turnoverthroughout adulthood, making it a useful modelto study neuronal development. Here we deter-mine the effect of PrPC level on neurodevelop-ment in two injury models: acute injury andprion-induced neurodegeneration.

Acute nasotoxic injury was induced bymethimazole injection and results in synchro-nized OSN regeneration. To investigate therole of PrPC in OSN proliferation, dividingcells in the OSE were quantified using BrdUincorporation. Gene expression indicative ofOSN differentiation was assessed by quantita-tive real-time PCR. Analysis revealed subtleeffects of PrPC on OSN differentiation andthese altered gene expression patterns offerpotential pathways to investigate PrPC functionin OSE neurogenesis. During prion infectionthere was an increase in nascent/immatureOSN differentiation markers and a reduction inmature OSN gene expression. Additionally, thenumber of neural progenitor cells was initiallyincreased, but over time fewer of those cellsremained in the OSE. These findings suggest aloss of mature OSNs during prion infection


either by premature death or a deficiency inOSN maturation, and as a result, there is anincrease in proliferation of neural progenitorcells to replenish the loss of mature OSNs.

O.17: Defining routes toneurodegeneration and the impact of the

immune system

Abigail Diack1, James Alibhai1,2,Lita Murphy1, James Vincenti1,Dot Kisielewski1, Aileen Boyle1,Pedro Piccardo1, V Hugh Perry3,Tom Freeman1, John Fazakerley4,

Rennos Fragkoudis4, andJean Manson1

1The Roslin Institute; University of Edinburgh;

Easter Bush UK; 2The National CJD Research &

Surveillance Unit; University of Edinburgh;

Edinburgh UK; 3University of Southampton;

Southampton UK; 4The Pirbright Institute;

Woking UK

In diseases such as Alzheimer’s disease orprion diseases, aberrant folding of host encodedproteins acts as seeds for ‘prion-like” propaga-tion of normally folded protein to abnormalconformations. Misfolded proteins are regardedas causal factors of disease although their pre-cise role in neurodegeneration remains unan-swered. Activation of glial cells is an early

pathological sign of disease, thus alterations inthe status of these cells in the brain may impacton the disease process. Indeed viral infectionhas a controversial role in chronic neurodegen-eration and has been implicated in both precipi-tating and driving the disease process.

Here we use prion models of neurodegenera-tion to demonstrate that prion disease can beboth transmissible and non-transmissible. Weexamined the role of misfolded protein in theprocess of disease and in contrast to generallyaccepted selective spread of misfolded proteins,‘prion seeds’ were widespread, distributedindependently of neurodegeneration. Despitethis, neurodegeneration and inflammatoryresponses were restricted to specific brainregions thus demonstrating that a misfoldedprotein seed is insufficient to initiate a neurode-generative cascade.

We examined the role of glial cells and theimpact of viral infection on the disease processand observe that a single co-infection eventwith a neurotropic viral agent has the ability toalter multiple aspects of disease, regional tar-geting, neuronal survival, inflammatory profileand biochemical properties of the misfoldedprion protein. Such co-infection events cantherefore have long term consequences for dis-ease progression.

These data address the underlying mecha-nisms of neurodegeneration and identify newtherapeutic targets for neurodegenerativediseases.


Prion 2015 Poster Abstracts

P.01: Unusual case of sporadicCreutzfeldt-Jakob disease VV1

subtype

Timothy Wuerz1, Brian Appleby1, andAlberto Bizzi2

1Case Western Reserve / University Hospitals;

Cleveland, OH USA; 2Istituto Neurologico Carlo

Besta; Milan, Italy

Creutzfeldt-Jakob disease (CJD) ischaracterized by a variety of symptomsincluding rapidly progressive neurocognitivedecline, ataxic gait, and myoclonus. SporadicCJD (sCJD) has a median disease duration of 6months. Based on the prion protein gene(PRNP) codon 129 polymorphism and prionprotein type, seven different molecularsubtypes of sCJD have been identified (MM1,MM2-C, MM2-T, MV1, MV2, VV1, VV2).The VV1 subtype is the rarest molecularsubtype of sCJD (only about 1% of cases). Todate, reported cases of sCJD VV1 have beencharacterized by young age at disease onset, anincreased length of disease duration, andprogressive neurocognitive degeneration. Wedescribe a unique case of sCJD VV1 with olderage at disease onset and short illness duration.VV1 is a challenging subtype to diagnose andthis report adds further complexity andvariation to its clinical characterization.Although PRNP codon 129 polymorphism andprion protein type exert a large influence onclinical and neuropathologic phenomenologyin sCJD, other factors remain unknown andrequire further study.

P.02: A transfectant RK13 cell linepermissive to caprine scrapie prion

infection

Rohana Dassanayake1, KatherineO’Rourke1, Dongyue Zhuang2,

Thomas Truscott2, Sally Madsen-Bouterse1, and David Schneider1,2

1Washington State University; Pullman, WA USA;2US Department of Agriculture; Pullman, WA USA

Classical scrapie is a transmissiblespongiform encephalopathy that affectsdomestic goats and sheep. Animal bioassay canbe performed to assess scrapie infectivityassociated with caprine-origin tissues butincubation periods are long. No scrapiepermissive cell line is currently available tostudy caprine scrapie. Therefore, the goals ofthis study were to generate a rabbit kidneyepithelial cell line (RK13) stably expressingcaprine wild type PRNP (cpRK13) and to assessits permissiveness to brain-derived caprinescrapie prion propagation. The cpRK13 andplasmid control RK13 cells (pcRK13) wereincubated with scrapie brain inocula preparedfrom three wild type goats and two heterozygousgoats (GS127 goat, or an IM142 goat).Significant accumulation of PrPSc was detectedby ELISA in cpRK13 inoculated with wild typecaprine scrapie inocula (3/3) but not incubationwith the inocula derived from the heterozygousgoats (0/2). However, PrPSc accumulationlevels were improved in cpRK13 inoculatedwith ovinized transgenic mice (Tg338) passaged

S11

Prion, 9:S11–S99, 2015ISSN: 1933-6896 print / 1933-690X online

caprine scrapie samples when compared to theoriginal heterozygous caprine scrapie inocula.Accumulation of PrPSc in wild type caprinescrapie inoculated cpRK13 cells could also bedetected by scrapie immunohistochemistry.Western blot analysis revealed typical di-,mono- and un-glycosylated proteinase K-resistant PrPSc isoforms in wild type caprinescrapie inoculated cpRK13 lysate. Importantly,PrPSc accumulation was not detected insimilarly inoculated pcRK13 cells, whether byELISA, western blot, or scrapie immuno-histochemistry. Taken together, these findingssuggest that cpRK13 may be a useful model toassess caprine classical scrapie in brain tissues.

P.03: Visual art therapy in sporadicCreutzfeldt-Jakob disease: A case study

Rajeet Shrestha1, Barbara Trauger-Querry2,Athena Loughrin3, and Brian Appleby1

1Case Western Reserve University and University

Hospitals; Cleveland, OH USA; 2Hospice of the

Western Reserve; Cleveland, OH USA; 3Lou Ruvo

Center for Brain Health; Cleveland Clinic;

Cleveland, OH USA

We describe the diagnostic and treatmentutility of visual art therapy in a case of sporadicCreutzfeldt-Jakob disease. Visual art therapywas collected and compared longitudinallywith clinical and neuroimaging data over acourse of 5 months in an autopsy confirmedcase of sporadic Creutzfeldt-Jakob disease ofthe MM2-cortical subtype. The visual art treat-ment sessions and ensuing content was usefulin ascertaining neuropsychiatric symptoms dur-ing the course of her illness. Art therapy alsooffered a unique emotional and cognitive outletfor patient and family as her illness progressed.Patients and families affected by sporadicCreutzfeldt-Jakob disease may benefit from arttherapy despite the rapid and progressive natureof the illness. Art therapy can also be useful forthe assessment of patients with sporadic Creutz-feldt-Jakob disease by healthcare professionals.

P.04: Conduct of diagnosis in the case ofoccurrence of bovine spongiform

encephalopathy in Romania

Florica Barbuceanu1, Marion Simmons2,Melanie Chaplin2, Cristina Diaconu1,S Nicolae1, Stefania Raita3, C Belu3,

and G Predoi3

1Institute for Diagnosis and Animal Health;

Bucharest, Romania; 2Animal and Plant Health

Agency; EU-RL-TSE; Weybridge, UK; 3University

of Agronomic Sciences and Veterinary Medicine of

Bucharest - Faculty of Veterinary Medicine;

Bucharest, Romania

Bovine spongiform encephalopathy (BSE) isa fatal infectious disease neurodegenerative,caused by prions that affects mainly cattle,characterized by spongiform and vacuolarchanges in the central nervous system (CNS).Although in the last period of time, the appear-ance of the classic cases of BSE had fallen, isthe appearance of atypical BSE forms, in manycountries of Europe, USA, Japan and Canada.The paper presents the conduct of diagnosis forthe first cases of BSE in Romania, reported onthe basis of laboratory investigations, the rapidtest positive to bovine animals slaughtered nor-mally diagnosed after confirmatory tests carriedout by the National Reference Laboratory forTransmissible Spongiform Encephalopathies(NRL-TSEs) within the Institute for Diagnosisand Animal Health (IDAH). After carrying outtests for the differentiation of strains of BSEsamples sent to the European Union ReferenceLaboratory for the Transmissible SpongiformEncephalopathies (EU-RL-TSE) from the Ani-mal and Plant Health Agency (APHA) Wey-bridge, United Kingdom, were diagnosed 2cases of atypical BSE, type L.

S12 Prion 2015 Poster Abstracts

P.05: In search for a mammaliandisaggregase function involved in prion

propagation

Basant Abdulrahman, Sandi Nishikawa,Sampson Law, Amalia Rose, and

Hermann Schatzl

University of Calgary; Calgary, AB Canada

Prion diseases are a family of transmissiblefatal neurodegenerative disorders that affectboth human and animals. They are character-ized by conformational conversion of the nor-mal cellular prion protein (PrPc) into thedisease associated isoform PrPSc. Accumula-tion of PrPSc aggregates leads to brain damage.The fragmentation of the aggregates intosmaller infectious seeds is considered a require-ment for prion propagation, a process whichinvolves disaggregase function. The responsi-ble mammalian disaggregase was not yet iden-tified. The role of autophagy, a basic cellulardegradation machinery, has been addressed inprion diseases by us and others. Our data inmouse embryonic fibroblasts (MEFs) show thathomozygous knockout of the autophagy geneAtg5 blocking autophagic flux results in aninability to effectively propagate PrPSc. On theother hand, wild-type MEFs showed increasedlevels of the autophagy marker LC3-II whenthey started propagating PrPSc. These datademonstrate that a basal level of autophagy isrequired for initiating a primary prion infection.Recently, it became a subject of intensiveresearch how the endosomal sorting complexrequired for transport (ESCRT) system isinvolved in completion of autophagy. Our con-focal microscopy data showed that persistentlyinfected neuronal cells (ScN2a) transfectedwith Rab 7, 9 or 11 (ESCRT system members)harbor more PrPSc aggregates compared tothose transfected with wild-type constructs.Interestingly, a transient knockdown of autoph-agy using siRNA against Atg5 increasedPrPScaggregates compared to control treatedScN2a cells. Taken together, our data demon-strate a possible role for ESCRT and autopha-gic machineries as a disaggregase in PrPSc

formation.

P.06: Assessing mother to offspringtransmission of chronic wasting disease

using a transgenic mouse model

Kassandra Willingham, Erin McNulty,Kelly Anderson, Jeanette Hayes-Klug,Amy Nalls, and Candace Mathiason


Chronic wasting disease (CWD) is the trans-missible spongiform encephalopathy (TSE), offree-ranging and captive cervids (deer, elk andmoose). The presence of infectious prions inthe tissues, bodily fluids and environments ofclinical and preclinical CWD-infected animalsis thought to account for its high transmissionefficiency. Recently it has been recognized thatmother to offspring transmission may contrib-ute to the facile transmission of some TSEs.Although the mechanism behind maternaltransmission is not yet known, the extendedasymptomatic TSE carrier phase (lasting yearsto decades) suggests that it may have implica-tions in the spread of prions.

Placental trafficking and/or secretion in milkare 2 means by which maternal prion transmis-sion may occur. In these studies we explorethese avenues during early and late infectionusing a transgenic mouse model expressing cer-vid prion protein. Na€ıve and CWD-infecteddams were bred at both timepoints, and wereallowed to bear and raise their offspring. Milkwas collected from the dams for prion analysis,and the offspring were observed for TSE dis-ease progression. Terminal tissues harvestedfrom both dams and offspring were analyzedfor prions.

We have demonstrated that (1) CWD-infected TgCerPRP females successfully breedand bear offspring, and (2) the presence ofPrPCWD in reproductive and mammary tissuefrom CWD-infected dams. We are currentlyanalyzing terminal tissue harvested from off-spring born to CWD-infected dams for thedetection of PrPCWD and amplification compe-tent prions. These studies will provide insightinto the potential mechanisms and biologicalsignificance associated with mother to off-spring transmission of TSEs.

Prion 2015 Poster Abstracts S13

P.07: The environmental neurotoxicantmanganese promotes prion-like cell-to-cell transmission of a-synuclein viaexosomes in cell culture and animal

models of Parkinson’s disease

Dilshan Harischandra, Matthew Neal,Arthi Kanthasamy, Nikhil Panicker,

Hope Privitera, Huajun Jin,Vellareddy Anantharam, andAnumantha Kanthasamy

Iowa State University; Ames, IA USA

The aggregation of a-synuclein (aSyn) isconsidered a key pathophysiological feature ofParkinson’s disease (PD). Recent studies sug-gest that a prion-like cell-to-cell transfer of mis-folded aSyn contributes to the spreading ofaSyn pathology. However, biological mecha-nisms underlying the propagation of the diseasewith respect to environmental neurotoxic chem-ical exposures not well understood. Consideringrole of the divalent metal manganese in PD-likeneurological disorders, we characterized itseffect on aSyn misfolding and protein aggrega-tion. First, we established an MN9D dopami-nergic cell line stably expressing wild-typehuman aSyn and treated it with non-toxic dosesof manganese at multiple time points. Analysisof condition medium (CM) through Westernblot showed that cells secreted aSyn into extra-cellular media following manganese exposure.Further characterization of CM through elec-tron microscopy readily detected nano-sizedvesicles with the characteristic hallmarks ofexosomes. Western blot and ELISA studiesrevealed that the exosomes do in fact containaSyn. Furthermore, Nanosight particle analysisshowed that manganese exposure enhances therelease of aSyn-containing exosomes. In func-tional studies, we demonstrated that exosomesreleased during manganese treatment caninduce neuroinflammatory responses in primarymicroglial cultures and neurodegeneration indifferentiated human dopaminergic cells(LUHMES) through the activation of caspase-3signaling. We also showed for the first time thatstereotaxic delivery of aSyn-containing

exosomes isolated from manganese-treatedaSyn-expressing cells into the striatum can ini-tiate PD-like motor deficits in mice. Collec-tively, these results demonstrate thatmanganese exposure promotes aSyn secretionvia exosomal vesicles, which subsequentlyevoke pro-inflammatory and neurodegenerativeresponses in both cell culture and animalmodels.

P.08: Two Korean families withGerstmann-Str€aussler-Scheinker disease

Sang-Beom Kim1, Yong-Sun Kim2, andJae Woo Kim3

1Department of Neurology; Kyung Hee University

Hospital at Gangdong; Seoul, Republic of Korea;2Ilsong Institute of Life Science; College of

Medicine; Hallym University; Seoul, Republic of

Korea; 3Department of Neurology; Dong-A

University Hospital; Busan, Republic of Korea

Gerstmann-Str€aussler-Scheinker disease(GSS) is a type of human transmissible spongi-form encephalopathy that is determined geneti-cally. We report 2 Korean families with GSS.

Case 1 A 46-year-old woman was admitteddue to a slowly progressive ataxic gait andspeech disturbance that had started 3 y earlier.On examination, she revealed abnormal Tan-dem gait and slight dysmetria. Cognitivedecline developed one year prior to admission.She had been bedridden for the 2 months priorto her admission. In the family history, 2 sistersout of 7 siblings developed similar symptomsin their fourth and fifth decade, respectively,and both expired approximately 5 y after theonset of symptoms. Case 2 A 31-year-oldwoman presented with unsteady gait, dysarthriaand dizzy sense which gradually progressed.Six months later, she could not walk indepen-dently. At the same time, severe memoryimpairment and incoherent speech werenoticed. No later than one month, she was bed-ridden and could respond only with simplewords. Her mother showed similar features and5 family members over 4 generation were sus-pected to have similar conditions. Both cases


revealed high signal intensities over entire cor-tex in diffusion weighted imaging. PRNP anal-ysis revealed a mutation in codon 102 prolineto leucine.

Conclusions. GSS should be considered fordifferential diagnosis when hereditary cerebel-lar ataxia and progressive cognitive declinedevelops. Unlike CJD, GSS is characterized bylong periods of illness, and dementia developslate in the course of the illness. The utilizationof diffusion-weighted MRI is suggested for theearly diagnosis of GSS.

P.09: LPS-induced systemicinflammation in goats naturally devoid

of prion protein

Øyvind Salvesen, Malin R Reiten,Arild Espenes, Michael A Tranulis,

and Cecilie Ersdal

Faculty of Veterinary Medicine and Biosciences;

Norwegian University of Life Sciences;

Oslo, Norway

Although a large body of knowledge supportthe obligate function of the prion protein (PrP)in human and animal prion disorders, evidenceof the normal function of PrP remains elusive.However, expression across tissues in mamma-lian species suggests that it may be involved ina variety of physiological functions. Recently,a nonsense mutation blocking the expression ofPrP in healthy Norwegian Dairy Goats was dis-covered as the first report of naturally-occur-ring PrP-free animals.

To investigate the role of PrP in the inflam-matory response, we plan a case-control studyof lipopolysaccharide (LPS)-induced inflamma-tion in homozygote (PRNPTer/Ter) goats carry-ing this mutation, compared to normal goats(PRNPC/C) expressing PrP. In a preliminarystudy, 2 PRNPC/C goats and one PRNPTer/Ter

goat were intravenously challenged with LPS(Escherichia coli O26:B6) twice. TwoPRNPC/C goats received corresponding vol-umes of saline. Clinical and behavioral parame-ters were monitored for 30 hours and blood

samples were collected during the experiment.After euthanization, tissue samples for histopa-thology, freezing and expression analysis werecollected.

Characteristic clinical signs of acute sepsisaccompanied by marked decrease of peripheralleukocytes were observed after LPS adminis-tration. Fever was most prominent in thePRNPTer/Ter goat. In neurons of hippocampaldentate gyrus, single cell necrosis were evidentin animals receiving LPS. Lung edema and sub-pleural bleedings were observed in thePRNPTer/Ter goat, microscopically accompa-nied by neutrophil leukostasis, increasedamount of alveolar macrophages and alveolarbleedings.

Our initial findings support that this LPSmodel is suitable for further work elucidatingPrP functions.

P.10: Recapitulation of prion diseasepathology and abnormal PrP depositionpatterns in organotypic cerebellar slices

Hanna Wolf1, Andrea Grassmann1,Andr�e Hossinger1, Lydia Paulsen1,

and Ina Vorberg1,2

1German Center of Neurodegenerative Diseases;

Bonn, Germany; 2Department of Neurology;

Rheinische Friedrich-Wilhelms-University;

Bonn, Germany

Organotypic cerebellar slices exhibit largelypreserved tissue architecture and represent asuitable model for characterizing and manipu-lating prion replication in a complex cell envi-ronment. However, the cellular distribution ofdisease specific PrPd in organotypic slices hasnot been assessed. Here we report the simulta-neous detection of disease-specific prion pro-tein PrPd and CNS markers in cerebellar slicesof C57BL/6 pups infected with mouse-adaptedprion strain 22L. Despite substantial abnormalPrP accumulation, no significant decrease inviability or mitochondrial respiration wasobserved over the course of 11.5 weeks. Still,epifluorescence and confocal microscopy ofintact, unsectioned slices revealed key


hallmarks of TSE pathology, including focalloss of purkinje cell dendrites, spongiformdegeneration in the molecular layer, and reac-tive astro- and microgliosis. Unmasking of PrPepitopes allowed for the specific detection ofpathologic PrP by immunocytochemistry. PrPd

distribution profiles in organotypic slicesclosely resembled those in vivo, demonstratinggranular spot like deposition predominately inthe molecular and purkinje cell layer. Doubleimmunostaining of PrPd and CNS cellularmarkers identified PrPd in the neuropil andassociated with astrocytes and microglia, butclear absence of PrPd in purkinje cells. Theestablished protocol for the simultaneousimmunocytochemical detection of PrPd andcellular markers in intact > 200 mm slices ena-bles detailed analysis on prion replication in acomplex ex vivo system.

P.11: Clinical features in Gerstmann-Str€aussler-Scheinker syndrome with

P105L mutation

Fumiko Furukawa1, Nobuo Sanjo1,Maya Higuma1, Tetsuyuki Kitamoto2,

Masaki Hizume1, Yoshikazu Nakamura3,Tadashi Yukamoto4, Shigeo Murayama5,Kagari Koshi6, Takashi Matsukawa6,

Shoji Tsuji6, Jun Goto6,Masahito Yamada7, Hidehiro Mizusawa2,

and Takanori Yokota1

1Tokyo Medical and Dental University; Tokyo,

Japan; 2Tohoku University Graduate School of

Medicine; Sendai, Japan; 3Jichi Medical

University; Tochigi, Japan; 4National Center of

Neurology and Psychiatry; Tokyo, Japan; 5Tokyo

Metropolitan Geriatric Hospital and Institute of

Gerontology; Tokyo, Japan; 6The University of

Tokyo Hospital; Tokyo, Japan; 7Kanazawa

University Graduate School of Medical Science;

Kanazawa, Japan

Introduction. Gerstmann-Str€aussler-Scheinker syndrome (GSS) with P105L muta-tion in the prion protein gene (PRNP; GSS105)has been reported only from Japan. The

Clinical features have not been clarifiedbecause of its very low predisposition.

Patient and Methods. We statistically ana-lyzed clinical symptoms and laboratory find-ings of GSS105 in the data collected byJapanese Prion Disease Surveillance betweenApril 1999 and September 2013, and comparedthem with those of GSS with P102L mutationin PRNP (GSS102).

Results. All 14 GSS105 cases, 8 males and6 females, had family history. Mean age atonset was 48.1, which was younger than that ofGSS102 (Kruskal-Wallis: p D 0.09). Most pre-dominant initial symptom was extrapyramidal(50%), followed by slowly progressive demen-tia (14%) and body pain (14%), and cerebellarsymptom was quite rare (7%). Ninety %, and75 % of the patients with GSS105 showedslowly progressive dementia and extrapyrami-dal symptoms, respectively. Periodic sharpwave complexs (PSWCs) were not detected inelectroencephalograms (EEGs) of all patients,even in patients showing myoclonus (36%).High intensity signal lesions in diffusion-weighted imaging of MRI were detected in14% of the patients. All the patients had129 MV heterozygosity, with the 129 V codonon the same allele as the 105 L.

Conclusion. Clinical feature of GSS105 wascharacterized by slowly progressive cognitiveimpairment and extrapyramidal signs withoutPSWCs in EEGs and with low frequency ofhigh signal intensity in MRI.


P.12: MALDI-TOF mass spectrometryfor analysis of polymorphisms in thegenome sheep for the detection of

susceptibility to scrapie

Daniele Macr�ı, Maurizio Bivona,Francesca Lo Mascolo,

Mariangela Colnago, Onofrio Buttitta,Gina Messina, and Fabrizio Vitale

Istituto Zooprofilattico Sperimentale della Sicilia;

Palermo, Sicily, Italy

The objective of this study was to comparethe field performances between 2 differentmethods, MALDI-TOF mass spectrometry andRT PCR for analysis of polymorphisms in thegenome sheep for the detection of susceptibilityto SCRAPIE. MALDI-TOF mass spectrometryis now being used for analysis of nucleic acids,including genetic variations such as microsatel-lites, insertion/deletions, and, especially, singlenucleotide polymorphisms (SNPs). The outputdata is a measure of an intrinsic characteristicof the DNA products being studied (molecularweight in Daltons); no indirect measurement ofthe products is involved, as with fluorescent orradiolabel tagging. The ability to resolve oligo-nucleotides varying in mass by less than a sin-gle nucleotide makes MALDI-TOF massspectrometry an excellent platform for SNPand mutation analysis. A highly automatedprocessing platform incorporating MALDI-TOF mass spectrometry, designated DNA Mas-sARRAYTM, has been developed. DNA Mas-sARRAYTM uses samples in chip-based,highdensity arrays. This system accurately callsSNPs in individual DNA samples, or alterna-tively determines SNP allele frequencies inDNA pools. Assay design for MassARRAYTMis simple, flexible and has been automated toallow designing vast numbers of assays, all ofwhich can be run using a universal set of reac-tion conditions.

P.13: Detection of sCJD prions in humansaliva by RT-QuIC

Matteo Manca1, Gianluigi Zanusso2, andByron Caughey1


Mountain Laboratories (RML); National Institute of

Allergy and Infectious Disease (NIAID); National

Institutes of Health (NIH); Hamilton, MT USA;2Department of Neurological and Movement

Sciences; University of Verona; Verona, Verona,

Italy

Sporadic Creutzfeldt-Jakob Disease (sCJD)is the most common form of human prion dis-ease. A recent study showed that prion seedingactivity is RT-QuIC-detectable in the olfactoryneuroepithelium of sCJD patients. Relativelyrapid turnover of the olfactory neuroepitheliumand nasal mucus clearance systems might leadto the transportation of prion seeds into the oralcavity and shedding through saliva. Pooledhuman saliva was spiked with sCJD prions andsubjected to different treatments to investigatethe suitability of such a sample as a new andnon-invasive diagnostic specimen. Our findingshighlighted the presence of yet unidentified fac-tor(s) that could lead to spontaneous conversionin the RT-QuIC assay. We will show our ongo-ing results on the attempt to identify the factor(s) and eliminate it/them.


P.14: Plant-derived vaccine againstchronic wasting disease using multimeric

deer PrPC and its derivatives asimmunogens

Joenel Alcantara1,2, Erin Brown1,Dalia Abdelaziz1, Yuzuru Taguchi1,

Sampson Law1, and Hermann M Schatzl1

1University of Calgary; Department of Comparative

Biology & Experimental Medicine, Faculty of

Veterinary Medicine, Calgary, AB Canada;2University of Calgary; Department of

Microbiology; Immunology & Infectious Diseases,

Calgary, AB Canada

Chronic wasting disease (CWD) is a priondisease that affects free-ranging and farmedcervids. It is the most contagious prion disease,transmissible with direct animal to animal con-tact or indirect exposure to prions in the envi-ronment. The prevalence of CWD hasincreased rapidly in North America and highinfection rates can be seen in captive and free-ranging cervids. Thus, it is apparent that thereis an urgent need to control and prevent thisdisease from spreading.

One potential means of controlling CWD isthrough active vaccination using cellular prionprotein (PrPC) as the immunogen. Moreover,one technology receiving attention is the use ofplant-based expression systems to producerecombinant proteins as vaccines. This plat-form provides an economical and viable alter-native to other vaccine production systemssuch as microbial and cell culture systems.Plants act as natural bioreactors with infrastruc-ture for cultivation and harvesting readily avail-able. Vaccines can stably accumulate in seedplants such as Arabidopsis thaliana and Bras-sica napus (Canola) and the seeds can be trans-ported and stored at ambient temperaturewithout refrigeration.

Our strategy is to produce a plant-derivedvaccine using cervid multimeric PrPC. Our pre-liminary results show that plant-derived deerPrPC multimers can stably accumulate in seedsof A. thaliana plants. To our knowledge this isthe first report of expression of recombinant

PrPC in a plant system. Several transgenic linesare currently being scaled-up for biochemicalanalysis and immunization experiments. Ouroverall goal is to develop an effective vaccineagainst CWD and prion-related diseases.

P.15: Characterization of differentialproteome expression of MM1 and VV2subtypes of sporadic Creutzfeldt-Jakob

disease (sCJD) in Cerebellum

Waqas Tahir1, Saima Zafar1,Matthias Schmitz1, Franc Llorens1,

Aman Deep Arora1, Neelam Younas1,Isidre Ferrer2, and Inga Zerr1

1Clinical Dementia Center, Department of

Neurology and Psychiatry, University Medical

Center Goettingen (UMG), Goettingen, Lower

Saxony, Germany; 2Institute of Neuropathology,

IDIBELL-University Hospital Bellvitge,

University of Barcelona, Hospitalet de Llobregat,

Barcelona, Spain

Keywords. neuro-degeneration, prion diseases,sporadic Creutzfeldt-Jakob disease,

Sporadic Creutzfeldt-Jakob disease (sCJD)is an example of human prion diseases with idi-opathic in its origin1. Codon 129 genotype ofPrP plays an important role in determining thesusceptibility of different parts of the brain toneuro-pathological events in sCJD2. VV2 sub-type shows an elevated expression of total andun-glycosylated PrP protein, distinct PrP pla-que-like deposition and IL-6 and TNF-a atmRNA level as compared to MM1 subtypeonly in Cerebellum part of the brain. Anincreased expression of GFAP along with vari-able expression of microglia in both subtypesMM1 and VV2 of sCJD and more micro-vacuoles based spongiform degeneration inMM1 subtype is seen in the cerebellum ofsCJD3. In this study, we aimed to characterizedifferential proteome expression of MM1 andVV2 subtypes of sCJD in Cerebellum part ofthe brain by using 2-Dimensional gel electro-phoresis. We had found total of 38 regulatedprotein spots between MM1 and control


samples, 43 regulated protein spots betweenVV2 and control samples and only 9 regulatedprotein spots between MM1 and VV2 samples.Further identification of these differentiallyregulated protein spots by Q-TOF MS/MS andthen validation of identified regulated proteinsby western blot was done. Altogether, theseresults indicate the codon 129 genotype of PrPbased proteomic alterations and role of PrPC inneurodegeneration in prion diseases in generaland in sporadic Creutzfeldt-Jakob disease inparticular which may help to discover someearly novel diagnostic markers and therapeuticstrategies as well.

P.16: Glycosaminoglycan modulationaffects cellular prion replication

downstream of PrPSc internalization

Hanna Wolf1, Catharina Pleschka1,Andrea Grassmann1, Romina Bester2,Andr�e Hossinger1, Christoph Moehl1,Lydia Paulsen1, Martin Groschup3,

Hermann Schaetzl4, and Ina Vorberg1,5

1German Center of Neurodegenerative Diseases;

Bonn, Germany; 2Institute of Virology; Technical

University of Munich; Munich, Germany;3Friedrich-Loeffler-Institut; Institute for Novel and

Emerging Infectious Diseases; Greifswald-Isle of

Riems, Germany; 4Faculty of Veterinary Medicine;

Dept. of Comparative Biology & Experimental

Medicine; University of Calgary; Calgary, Canada;5Department of Neurology; Rheinische Friedrich-

Wilhelms-University; Bonn, Germany

Prions are unconventional infectious agentscomposed primarily of misfolded aggregatedhost prion protein PrP, termed PrPSc. Conver-sion of cellular prion protein into PrPSc occurson the cell surface or along the endocytic path-way. The precise mechanisms and cellularrequirements for PrPSc uptake, the initial PrPSc

formation and the persistent PrPSc propagationstill remain unknown. Glycosaminoglycans(GAGs), highly-sulfated unbranched polysac-charides present on the cell surface and withinendocytic vesicles, have been implicated as firstattachment sites for prions and cofactors for

replication. GAG mimetics and obstruction ofGAG sulfation affect prion replication, but sofar, comparative analysis of the role of GAGsduring the individual stages of infection by 22Lprion strain has not been performed. We exam-ined the effect of the GAG mimetic, DS-500,and the sulfation inhibitor, NaClO3, on prioninfection by scrapie strain 22L in L929 cellsand organotypic cerebellar slices. Here weshow that both compounds change the cellulardistribution and levels of sulfated GAGs buthave divergent effects on cell surface and totalPrPC levels in L929 cells. Chemical manipula-tion of GAGs did not prevent PrPSc uptake,arguing against their role as essential attach-ment sites. Importantly, GAG undersulfationand DS-500 effectively antagonized de novoand chronic 22L prion infection in L929 cellsand organotypic cerebellar slices. We concludethat DS-500 and NaClO3 affect events down-stream of the initial PrPSc attachment andinternalization.

P.17: High throughput detection of PrPSc

from prion-infected cells without PKtreatment: Cell-based ELISA for novel

screening method for anti-prioncompounds

Zhifu Shan, Takeshi Yamasaki,Akio Suzuki, Rie Hasebe, and

Motohiro Horiuchi

Laboratory of Veterinary Hygiene; Graduate

School of Veterinary Medicine; Hokkaido

University; Sapporo, Japan, China

Screening of chemical libraries is one of thepossible ways for identification of therapeuticcompounds for prion diseases. Prion-infectedcells are often used for analyzing the effect ofcompounds on PrPSc formation. Mostly, PrPSc

is detected by anti-PrP antibodies after aremoval of PrPC by proteinase K (PK) treat-ment. However, PK-sensitive part of PrPSc

(PrPSc-sen) that possesses higher infectivityand conversion activity than the PK-resistantPrPSc (PrPSc-res) is expected to be also digestedby PK treatment. To overcome this problem, in


this study, we attempted to establish a novelcell-based ELISA for screening of anti-prioncompounds, in which PrPSc can be directlydetected from prion-infected cells without PKtreatment. The mAb 132 that recognizes epi-tope consisting of mouse PrP aa 119–127enabled us to detect PrPSc directly from prion22L strain-infected Neuro2a cells (N2a) pre-treated with GdnSCN without PK treatment.PrPSc also could be detected from Chandlerstrain-infected N2a cells and 22L strain-infectedGT1–7 cells. Analytical dynamic range forPrPSc detection was about 1Log. The coefficientof variation and signal to background ratio were711%– and 2.5–3.3, respectively, demonstratingthe reproducibility and stability of this assay.The addition of WST assay for evaluating cyto-toxicity of compounds did not influence the fol-lowing PrPSc detection, therefore, all theprocedures including culture, cytotoxicity assayand PrPSc detection can be completed in thesame plate. The simplicity and no requirementfor PK treatment of the novel cell-based ELISAappear to be remarkable advantages for highscreening of anti-prion compounds.

P.18: Attempts to amplify Nor98 in vitroby bank vole PMCA

Ilaria Vanni, Laura Pirisinu,Michele Angelo Di Bari,

Claudia D’Agostino, Umberto Agrimi,and Romolo Nonno

Department of Food Safety and Veterinary Public

Health; Istituto Supeirore di Sanit�a; Rome, Italy

Despite PMCA was reported to replicateseveral prion strains, the in vitro amplificationof TSEs characterized by atypical PrPSc, withprotease-resistant core cleaved at both the Nand C-termini, hasn’t been reported so far. Werecently showed that bank voles carrying iso-leucine at PrP residue 109 (Bv109I), but notthose carrying methionine (Bv109M), arehighly susceptible to Nor98 and GSS, bothcharacterized by atypical PrPSc, suggesting acentral role of host PrPC sequence in the per-missiveness to these TSEs. We aimed at

investigating if this principle operates in vitrotoo, by attempting to replicate in vitro Nor98by PMCA using brain substrates from Bv109Mand Bv109I.

In PMCA reactions performed with our stan-dard protocol, no amplification was observed inboth substrates using either ovine or vole-adaptedNor98 seeds. Although a PrPres signal decreasewas occasionally observed in samples subjectedto PMCA, further experiments showed that nei-ther continuous sonication up to 5 minutes norincubation at 90�C affected the stability of Nor98PrPSc. Negative results were also obtained bychanging the length of PMCA cycles, the deter-gents or by adding Teflon beads in PMCA tubes.

The apparent lack of amplification mightsimply represent a feature of Nor98, as differ-ent prion strains show different replication effi-ciencies in PMCA. Alternatively, atypicalPrPSc itself might be unable to induce in vitroaggregation of PrPC, supposedly because ofpeculiar mechanisms of aggregation not sup-ported by PMCA. To investigate this hypothe-sis we are now testing other TSEs with atypicalPrPSc for their ability to replicate in PMCA.

P.19: Characterization of chronicwasting disease isolates from free-

ranging deer (Odocoileus sp) in Albertaand Saskatchewan, Canada

Camilo Duque Velasquez1, Chiye Kim1,Nathalie Daude1, Jacques van der Merwe1,

Allen Herbst1, Trent Bollinger2,Judd Aiken1, and Debbie McKenzie1

1Centre for Prions and Protein Folding Diseases;

University of Alberta; Edmonton, Canada; 2Western

College of Veterinary Medicine; University of

Saskatchewan; Saskatoon, Canada

Chronic wasting disease (CWD) is anemerging prion disease of free ranging and cap-tive species of Cervidae. In North America,CWD is enzootic in some wild cervid popula-tions and can circulate among different deerspecies. The contagious nature of CWD prionsand the variation of cervid PRNP alleles, whichinfluence host susceptibility, can result in the


emergence and adaptation of different CWDstrains. These strains may impact transmissionhost range, disease diagnosis, spread dynamicsand efficacy of potential vaccines. We are char-acterizing different CWD agents by biochemi-cal analysis of the PrPCWD conformers,propagation in vitro cell assays1 and by com-paring transmission properties and neuropathol-ogy in Tg33 (Q95G96) and Tg60 (Q95S96)mice.2 Although Tg60 mice expressing S96-PrPC have been shown resistant to CWD infec-tivity from various cervid species,2,3 thesetransgenic mice are susceptible to H95C CWD,a CWD strain derived from experimental infec-tion of deer expressing H95G96-PrPC. Thediversity of strains present in free-ranging muledeer (Odocoileus hemionus) and white-taileddeer (Odocoileus virginianus) from Albertaand Saskatchewan is being determined and willallow us to delineate the properties of CWDagents circulating in CWD enzootic cervid pop-ulations of Canada.

References1. van der Merwe J, Aiken J, Westaway D, McKenzie D.

The standard scrapie cell assay: Development,

utility and prospects. Viruses 2015; 7(1):180–198;

PMID:25602372; http://dx.doi.org/10.3390/v7010180

2. Meade-White K, Race B, Trifilo M, Bossers A, Favara

C, Lacasse R, Miller M, Williams E, Oldstone M,

Race R, Chesebro B. Resistance to chronic wasting

disease in transgenic mice expressing a naturally

occurring allelic variant of deer prion protein. J Virol

2007; 81(9):4533–4539; PMID: 17314157; http://dx.

doi.org/10.1128/JVI.02762-06

3. Race B, Meade-White K, Miller MW, Fox KA, Chese-

bro B. In vivo comparison of chronic wasting disease

infectivity from deer with variation at prion protein

residue 96. J Virol 2011; 85(17):9235–9238; PMID:

21697479; http://dx.doi.org/10.1128/JVI.00790-11

P.20: Efficient in vivo propagation of PrPamyloids in transgenic mice expressing

anchorless prion proteins

Jan Sto€hr1,2, Carlo Condello1,2,Abby Oehler1,3, Stephen DeArmond1,3,

Holger Wille1,2, Gerald Stubbs4,Kurt Giles1,2, and Stanley Prusiner1,2

1Institute for Neurodegenerative Diseases;

University of California San Francisco; San

Francisco, CA USA; 2Department of Neurology;


Francisco, CA USA; 3Department of Pathology;


Francisco, CA USA; 4Vanderbilt University;

Nashville, TN USA

PrPSc displays structural diversity, rangingfrom small oligomers to large aggregates. Itwas shown that non-amyloidogenic PrP prionstrains could be converted into amyloidogenicprions by inoculation into transgenic miceexpressing anchorless PrP [Tg(PrP*DGPI)].Following inoculation of Tg(PrP*DGPI) micewith RML prions, we observed a substantialshortening of the incubation periods upon serialpassaging. This was accompanied by the accu-mulation of fold5- more PK-resistant, anchor-less PrP in second and third passages.Following these passages, anchorless PrPprions were passaged into mice expressing wtPrPC. Although still transmissible, we observedthat the prion titer dropped »104 ID50 units,suggesting that only a small fraction of anchor-less PrP was converted into PrPSc structurewhile most of the anchorless PrP assembledinto amyloid fibrils that were distinct fromPrPSc. Furthermore, inoculation of amyloido-genic recombinant (rec) PrP (residues 23–231and 89–230) into Tg(PrP*DGPI) mice alsoresulted in signs of neurologic dysfunctionwithin 200–300 d postinoculation. Serial trans-mission of synthetic, anchorless PrP prions inTg(PrP*DGPI) mice further shortened the incu-bation time. Compared to GPI-anchored, RMLprions upon primary passage, incubation timesfor anchorless prions in Tg(PrP*DGPI) micewere 200–300 d shorter. We suggest that thisdifference in the susceptibility of Tg


(PrP*DGPI) mice to prions derived from recPrPamyloids and natural sources such as sheepscrapie is due to structural differences thatwere found in fiber diffraction studies. In sum-mary, our studies demonstrate the profound dif-ferences in biological properties of prions withor without a GPI anchor.

P.21: Structural analysis of theanti-scrapie activity of DB772 in a

persistently-infected ovine microgliaculture system

Kelcey Dinkel1, David Schneider2,David Boykin4, Chad Stephens5,Sally Madsen-Bouterse1, and

James Stanton1,3

1Veterinary Microbiology and Pathology;

Washington State University; Pullman, WA USA;2United States Department of Agriculture;

Agricultural Research Service; Animal Disease

Research Unit; Pullman, WA USA; 3Department of

Pathology; University of Georgia; Athens, GA USA;4Department of Chemistry; Georgia State

University; Atlanta, GA USA; 5Department of

Chemistry and Physics; Georgia Regents

University; Augusta, GA USA

Despite efforts to identify anti-prion com-pounds, an effective therapeutic does not exist.The in vitro inhibitory activity of several com-pounds has failed to translate in vivo, whichmay be attributed to use of systems adapted tolab animals. The purpose of this study was todetermine the structural basis for the anti-prionactivity of a novel, monocationic phenyl-furan-benzimidazole (DB772) by screening an exist-ing library of related structure-substituted com-pounds in a scrapie microglial cell culturesystem. Eighty-9 compounds, includingDB772, were tested at 1mM in human telome-rase-immortalized ovine microglia. Anti-prionactivity was determined by the Standard Scra-pie Cell Assay. The effects of compounds oncell growth and PrPC content were respectivelyidentified by WST-1 assay and ELISA. Theeffect of DB772 on PrPSc-seeded misfoldingwas determined by PMCA.

Thirteen compounds were considered tootoxic for further study. Seven compounds werenontoxic and reduced PrPSc accumulation by� 1/2-log. Anti-prion activity appeared depen-dent on a central 5-membered heterocyclicring. Activity in such molecules was variablyaffected by the combinations of benzimidazoleand imidazole side groups, and terminalamidines. The greatest anti-prion activity wasassociated with symmetrical bis-benzimidazolestructures containing a 5-membered heterocy-clic aromatic ring. Active compounds did notappear to affect total PrPC. Preliminary resultsusing DB772 show concentration-dependentinhibition of PrPSc-seeded misfolding. In con-clusion, insights into the structural determi-nants of the anti-scrapie activity of DB772-related compounds were identified. Activecompounds did not appear to effect total cellu-lar content of PrPC, but may act by reducingconversion of PrPC to PrPSc.

P.22: Conformational dynamics of thecellular prion protein

Patricia Soto1, Natalie Bui2,Margaret Carter3, Roger Gonzales3,

William Graft4, Angela Lax3, Chad Nieri3,Bo Zhao1, and Jason Bartz5

1Physics Department; Creighton University;

Omaha, NE USA; 2Chemistry Department;

Creighton University; Omaha, NE USA; 3Biology

Department; Creighton University; Omaha, NE

USA; 4Mathematics Department; Creighton

University; Omaha, NE USA; 5Medical

Microbiology and Immunology Department;

Creighton University; Omaha, NE USA

Prions are infectious agents responsible fortransmissible spongiform encephalopathies, afatal neurodegenerative disease in mammals,including humans. Prions propagate biologicalinformation by conversion of the nonpathologi-cal version of the prion protein, PrPc, to theinfectious conformation, PrPSc. As a first stepto decipher the prion conversion mechanism,we aim at understanding the equilibrium con-formational dynamics of the cellular isoform.


We will report on structural bioinformatics andmultiscale molecular modeling studies to inves-tigate the interplay between PrPc topology andfragment amylogenicity; and the modulation onthe observed behavior by the local milieu, i.e.bulk vs. cell surface.

P.23: An improved test for the detectionof Creutzfeldt-Jakob Disease fromhuman CSF using new RT-QuIC

conditions

Bradley R Groveman1, Christina D Orr�u1,Andrew G Hughson1, Gianluigi Zanusso2,Maurizio Pocchiari3, Michael B Coulthart4,

and Byron Caughey1


Mountain Laboratories; NIAID; NIH; Hamilton,

MT USA; 2Department of Neurological and

Movement Sciences; University of Verona; Verona,

Italy; 3Department of Cell Biology and

Neurosciences; Istituto Superiore di Sanit�a; Rome,Italy; 4Canadian CJD Surveillance System; Public

Health Agency of Canada; Ottawa, ON Canada

Neurodegenerative protein misfolding dis-eases are difficult to diagnose early and accu-rately. This is particularly worrisome withhuman prion diseases, such as Creutzfeldt-Jakob disease (CJD), because prions are trans-missible, deadly, and unusually resistant todecontamination. Real-time quaking-inducedconversion (RT-QuIC) assays allow highly sen-sitive and specific testing for CJD using humancerebrospinal fluid (CSF) or nasal brushingsand are being widely implemented as importantdiagnostic tools. However, such laboratoryanalyses have required 2.5 to 5 d to complete.Furthermore, CSF testing using previouslyevaluated RT-QuIC conditions still yields falsenegative results in 11 to 23% of CJD cases. Wehave now developed an improved RT-QuICassay which can identify positive CSF sampleswithin 4 to 14 h with better analytical and diag-nostic sensitivity. Analysis of CSF samplesfrom 11 CJD patients demonstrated that while7 were RT-QuIC positive using previous condi-tions, an additional 3 samples were positive

using the new assay. In these and subsequentanalyses, a total of 46 of 48 CSF samples fromsporadic CJD patients gave positive RT-QuICresponses, while all 39 non-CJD patients werenegative, giving 95.8% diagnostic sensitivityand 100% specificity. This diagnostic sensitiv-ity was significantly better than that obtainedusing the previous conditions. We continue toexpand the testing of CJD-positive and -nega-tive CSF samples to further establish the diag-nostic utility of this new assay for varioushuman prion diseases. So far, our improvedRT-QuIC assay appears to allow for muchfaster, more accurate and practical antemortemtesting for CJD using CSF samples.

P.24: A comparative study of dura matergraft-associated Creutzfeldt-Jakobdisease between Japan and other

countries

Tsuyoshi Hamaguchi1, Kenji Sakai1,Moeko Noguchi-Shinohara1,

Ichiro Nozaki1, Ichiro Takumi2,Nobuo Sanjo3, Yosikazu Nakamura4,Tetsuyuki Kitamoto5, Nobuhito Saito6,

Hidehiro Mizusawa7, andMasahito Yamada1

1Department of Neurology and Neurobiology of

Aging; Kanazawa University Graduate School of

Medical Science; Kanazawa, Japan; 2Department

of Neurosurgery; Nippon Medical School Musashi

Kosugi Hospital; Kawasaki, Japan; 3Department of

Neurology and Neurological Science; Graduate

School; Tokyo Medical and Dental University;

Tokyo, Japan; 4Department of Public Health; Jichi

Medical University; Shimotsuke, Japan;5Departments of Prion Protein Research; Division

of CJD Science and Technology; Tohoku University

Graduate School of Medicine; Sendai, Japan;6Department of Neurosurgery; Faculty of Medicine;

The University of Tokyo; Tokyo, Japan; 7National

Center Hospital; National Center of Neurology and

Psychiatry; Tokyo, Japan

Objective. More than 60% of patients world-wide diagnosed with Creutzfeldt-Jakob disease


(CJD) associated with dura mater graft (dCJD)have been identified in Japan. The remarkablefrequency of dura mater graft use in Japanmight contribute to the elevated incidence ofdCJD, but the possible reasons for the dispro-portionate use of this procedure in Japanremain unclear. We investigated the differencesbetween dCJD patients in Japan and those else-where to help uncover an explanation for theunusually more frequent use of cadaveric duramater and high incidence of dCJD in Japan.

Methods. We obtained data of dCJD patientsin Japan from the nationwide surveillance ofCJD in Japan and of those in other countriesfrom extant literature. We compared demo-graphic, clinical, and pathological features ofdCJD patients between Japan and elsewhere.

Results. Data from 142 dCJD patients inJapan and 53 in other countries were obtained.The medical conditions precipitating duramater graft were significantly different betweenJapan and other countries (P < 0.001); inJapan, there were more cases of cerebrovascu-lar disease and hemifacial spasm or trigeminalneuralgia. Patients with dCJD in Japan receiveddura mater graft more often for non-life-threat-ening conditions, such as meningioma, hemifa-cial spasm and trigeminal neuralgia, than thosein other countries.

Conclusion. Differences in the medical con-ditions precipitating dura mater graft may con-tribute to the frequent use of cadaveric duramater and the higher incidence of dCJD inJapan.

P.25: Subcutaneously administered LPS-converted recombinant mouse prionprotein alone or in combination with

LPS modulates the content ofprion-related proteins in the brain

of FVB/N mice

Grzegorz Zwierzchowski,Dagnachew Hailemariam, Suzanna Dunn,

David Wishart, and Burim Ametaj

University of Alberta; Edmonton, AB Canada

We tested whether subcutaneous (sc) injec-tion of LPS-converted mouse moPrP, which isresistant to proteinase K digestion (moPrPres),was able to affect concentrations of prion-relatedproteins including PRNP, SPRN, and PRND inthe brains of wild type FVB/N female mice. Wealso explored the effect of using moPrPres aloneor in combination with E. coli LPS. Five groupsof mice (n = 15) were treated sc either withsaline (CONtrol) or lipopolysaccharide (LPS),or with PrPres, PrPres plus LPS, RML, and RMLplus LPS. Saline and LPS were injected chroni-cally for 6 weeks by ALZET minipumps;whereas PrPres and RML were administered as aone time injection at the beginning of the experi-ment. Mice were allowed to develop neurode-generative disease and euthanized at theterminal stage. Brain homogenates were ana-lyzed with ELISA kits to measure the concentra-tions of PRNP, SPRN, and PRND. Resultsshowed that all treatments increased concentra-tions of PRNP with moPrPres increasing it morethan fold3- and RML almost fold5-. Combina-tions of moPrPres with LPS and RML with LPS,or LPS alone, elevated concentrations of PRNPversus saline. In addition, moPrPres increasedSPRN almost fold6- and treatment withmoPrPres and LPS more than fold15- comparedto CON. Treatment with RML increased SPRN2.fold8-, whereas combination of RML withLPS enhanced the amount of SPRN 1.fold4- vssaline. moPrPres increased PRND concentrationfold2- and RML fold3- vs CON. Additionally,moPrPres and RML with LPS and LPS aloneincreased concentrations of PRND in the brain


homogenates of mice compared to CONanimals.

P.26: Allele-specific RNA interference(RNAi) mitigates pathology in the Tg

(PrP-A116V) mouse model ofGerstmann-Str€aussler-Scheinker

disease (GSS)

Kefeng Qin, Crystal Gregory-Busch,Lili Zhao, Ani Solanki, and

James A Mastrianni

The University of Chicago; Chicago, IL USA

Allele-specific RNAi is an attractive thera-peutic approach for genetic prion diseases, as ittargets the pathogenic allele, thereby limitingpotential problems of long-term wild type (wt)PrP suppression. Our Tg(PrP-A116V) micemodel human GSS(A117V) and develop thecardinal features of progressive gait ataxia andPrP amyloid plaque accumulation, primarilywithin the cerebellum. We, therefore, designedsiRNAs differing in length (19 or 21 nucleoti-des) and position of the target mutationsequence (5’ end, mid-position, or 3’ end) andtested the selectivity and efficacy of each toinhibit expression of PrP-116V in transfectedCOS-7 and N2a cells, for eventual applicationin Tg(PrP-A116V) mice. A 19 nt siRNA withthe target sequence at the 3’ end displayed thebest profile. This was prepared as a short hair-pin (sh) RNA and incorporated into a lentiviralvector that was injected into the cerebellum of45 day-old Tg(PrP-A116V) mice. Comparedwith empty vector control (C), lentivirus carry-ing active (A) shRNA reduced cerebellar PrP-116V mRNA by 62.3 § 6.8% (p < 0.01) andPrP plaque burden by 72.2§ 2.2 % (P < 0.01),although disease onset [A D 130 .0§ 11.3 vs.C D 123 .2§ 16.1 d] and death [A D 164 .4§16.6 d vs. C D 159 .5 § 21 .3 d], were onlyslightly, but not significantly, delayed. Equiva-lent injections in wt mice did not affect wt-PrPmRNA. These results are encouraging, as thereduction of cerebellar histopathology predictsa likely clinical benefit with expanded CNS

expression of the shRNA. They also highlightthe importance of non-cerebellar brain regionsin the development of the clinical phenotype ofGSS.

P.27: Intracranial injection of LPS-converted mouse recombinant PrP alone

or with LPS causes brainneurodegeneration in FVB/N mice

Grzegorz Zwierzchowski, Nathalie Daude,Suzanna M Dunn, David Wishart, and

Burim Ametaj

University of Alberta, Edmonton, Alberta, Canada

Recently we reported that lipopolysaccha-ride (LPS) from Escherichia coli 0111:B4 isable to convert the mouse prion protein into ab-rich isoform resistant to proteinase K(moPrPres). We also tested whether intracranial(ic) administration of moPrPres (29–232) is ableto cause prion-like pathology in vivo. Sixty fiveFVB/N female mice were treated ic with 30 mLof: 1) 1% NBH (CON) 2) LPS (0.5 mg/mL), 3)moPrPres (0.5 mg/mL), 4) moPrPresCLPS(0.5 mg/mL each), and 5) 1% RML (107 IDunits). Thirteen animals died during ic, includ-ing 7 from LPS and 4 from RML treatment.Animals that survived ic injection were left todevelop clinical signs of disease until terminalsickness or terminated at 589 post inoculationwithout clinical signs. Hematoxilin and eosin(H&E) and PrPSc-stainings were conducted todetermine presence of vacuolation and PrPSc

accumulation in the brains, respectively. Alltreatment groups, except for saline, showedbrain vacuolation in the cerebral cortex (Cc),thalamus (Th), midbrain (Mb), and cerebellum(Cr) and mild PrPSc accumulation only in theRML group. Larger number of vacuoles werefound in Th and Cr regions of moPrPres,PrPresCLPS, and RMLCLPS treated mice vsthe control (CON) group. Greater neurodegen-eration of Cc white matter was observed inmoPrPres and PrPresCLPS vs RML group. ThePrPSc-staining showed PrPSc accumulation onlyin the RML group. In conclusion moPrPres,LPS, and PrPresCLPS treated mice developed


brain neurodegeneration at terminal sickness.Treatment with LPS and PrPresCLPS seems tocause larger vacuoles in the brain compared tothe RML-only group.

P.28: Proteins are recruited intopolyglutamine aggregates via theirintrinsically-disordered domains

Maggie Wear1, Robert O’Meally2,Dmitry Kryndushkin1, and

Frank Shewmaker1

1Uniformed Services University; Bethesda, MD

USA; 2Johns Hopkins University; Baltimore,

MD USA

Intracellular protein aggregation is the hall-mark of several neurodegenerative diseases.Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, assumeamyloid-like structures that are resistant todenaturation. We combined mass spectrometryand a stringent purification procedure to iden-tify the protein species that are trapped withinaggregates formed by Huntingtin N-terminalfragments with pathogenic polyQ tracts(> 40 glutamines) in both yeast and mamma-lian (PC12) cells. We found that protein qual-ity-control and RNA-binding proteins weregreatly enriched in polyQ aggregates, anddespite their evolutionary divergence, therewas significant conservation between trappedproteins identified from yeast and PC12 cells.Notably, in the mammalian cells, a number ofneurodegenerative disease-linked proteins wereconsistently found trapped in the polyQ aggre-gates. Many of these proteins are found in neu-ronal inclusions in their respective diseases,suggesting that polyQ aggregates can recruitproteins that are prone to aggregation in differ-ent pathological contexts. We also analyzed theprimary and secondary structure of our aggre-gate-associated proteins and discovered a sig-nificant enrichment of proteins with very longintrinsically-disordered (ID) domains. Whenwe truncated the ID domains of selected pro-teins, the proteins no longer co-aggregated withpolyQ. The high frequency of ID domains in

RNA-binding proteins may explain why theseproteins are disproportionately found in patho-logical inclusions in many neurodegenerativediseases.

P.29: Bile acids reduce prion conversionand neuronal death in models of prion

disease

Leonardo Cortez1,2, Jody Campeau1,2,Grant Norman1,2, Marian Kalayil1,2,

Jacques Van der Merwe1,Debbie McKenzie1,3, and Valerie Sim1,2,4


Edmonton, Alberta, Canada; 2Department of

Medicine; Division of Neurology; University of

Alberta; Edmonton, Alberta, Canada; 3Department

of Biological Sciences; University of Alberta;

Edmonton, Alberta, Canada; 4Neuroscience and

Mental Health Institute; University of Alberta;

Edmonton, Alberta, Canada

No treatment is available for prion diseases.The bile acids tauroursodeoxycholic acid(TUDCA) and ursodeoxycholic acid (UDCA)have been recently shown to be neuroprotectivein models of other protein misfolding diseaseslike Parkinson’s, Huntington’s and Alzheimer’sdiseases. We studied the therapeutic efficacy ofthese compounds in prion disease models. Wedemonstrated that TUDCA and UDCA substan-tially reduced PrP conversion in cell-freeaggregation assays as well as in chronicallyand acutely infected cell cultures. This effectwas mediated through a reduction of the seed-ing ability of PrPSc, rather than an effect onPrPC. We also demonstrated the ability ofTUDCA and UDCA to prevent neuronal loss inprion infected cerebellar slice cultures. In addi-tion, we found that the levels of the proteinPSD-95 were modulated by the treatment andthis modulation was independent from theinhibitory effect of bile acids on PrPSc accu-mulation. Lastly, we demonstrated that lowdoses of UDCA reduced the levels of GFAP-driven luciferase induced by prion infection inGFAP-luc mice and prolonged the lifespan ofinfected C57Bl6 mice. Interestingly, these


effects were limited to the males, implying agender-specific difference in drug metabolism.Thus, as demonstrated for other neurodegenera-tive diseases, we propose that TUDCA andUDCA may have a therapeutic role in priondiseases, with effects on both prion conversionand neuroprotection. Our findings, togetherwith the fact that these natural compounds areorally bioavailable, permeable to the blood-brain barrier and FDA-approved for use inhumans, make these compounds promisingalternatives for the treatment of prion diseases.

P.30: Identification of Q,N-richtranscription factors that increase thesynthetic lethality of prion [PSIC]

with sup45mutations inSaccharomyces cerevisiae

Andrei Matveenko2,3, Mikhail Belousov1,Svetlana Moskalenko1,2,

Anton Nizhnikov1,2, Yuri Barbitoff1,Polina Drozdova1,3, Stanislav Bondarev1,

and Galina Zhouravleva1,3

1Dept. of Genetics and Biotechnology;

St Petersburg State University; St Petersburg,

Russia; 2St Petersburg Branch; Vavilov Institute of

General Genetics of the Russian Academy of

Sciences; St Petersburg, Russia; 3Laboratory of

Amyloid Biology; St Petersburg State University;

St Petersburg, Russia

Previously, we proposed a synthetic lethalitytest for genes that may influence the propertiesof translation termination factors Sup35 andSup45. It is based on the fact that combinationof sup45 mutations with [PSIC] prion in dip-loids is fatal. Strong [PSIC]S variant, which is astrong suppressor, causes synthetic lethality incombination with all of the nonsense and somemissense sup45 mutations tested, while weak[PSIC]W or [psi¡] phenotypes do not lead todiploid cell death. The presence of extra copiesof a gene that affects the manifestation of[PSIC] or termination factors properties maylead to either increase or decrease in diploidlethality. To search for new genes that affectthe translation termination efficiency and/or

prion maintenance a gene library screen usingthe synthetic lethality test was performed. Weidentified several genes, including Q,N-richtranscription factor MCM1. We analyzed otherknown Q,N-rich transcription factors and dis-covered that the synthetic lethality alsoincreases under overexpression of GLN3,MOT3 and SFP1. We propose that Q,N-richtranscription factors may influence SUP35expression thus affecting [PSIC]. This mightrepresent novel pathway of prion-prion interac-tions, since 2 of the transcription factors areknown prion determinants. The research wassupported by RRC MCT SPbSU. The authorsacknowledge Saint-Petersburg State Universityfor a research grants 1.37.291.2015,0.37.696.2013 and Russian Foundation ofBasic Research for a research grants 13-04-00645 and 14-04-31265.

P.31: In vivo C2-fragmentation of thecellular prion protein in uninfected cells

Ghazaleh Eskandari-Sedighi1,4,Gerold Schmitt-Ulms2, Robert CC Mercer1,

Nathalie Daude1, Jan PM Langeveld3,Serene Wohlgemuth1, and

David Westaway1,4


University of Alberta; Edmonton, AB, Canada;2Tanz Centre for Research in Neurodegenerative

Diseases; Department of Laboratory Medicine and

Pathobiology; University of Toronto; Toronto, ON,

Canada; 3Department of Infection Biology; Central

Veterinary Institute part of Wageningen UR;

Lelystad, The Netherlands; 4Department of

Biochemistry; University of Alberta; Edmonton,

AB, Canada

The domain organization of the cellularprion protein (PrPC) consists of an unstructuredN-terminal region, including a metal-bindingoctarepeat region (OR), a linker domain, and aC-terminal domain that misfolds to form PrPSc

in diseases like Creutzfeldt-Jakob Disease.PrPCcan be alternatively processed; b-fragmen-tation that results in formation of C2 fragment,a-endoproteolysis and shedding. Also,


oligomeric A-Beta peptide has a major bindingsite between the alpha- and beta-sites of PrPC.

b-cleavage of PrPC has previously been sug-gested to occur through reactive oxygen spe-cies generated by the binding of copper tothe PrP OR. We have recently reported gen-eration of a novel Prnp allele, “S3” whichhas PrPC OR conformationally-locked in acompact arrangement that favors binding ofone copper ion per OR. Interestingly, thisallele resulted in overproduction of C2 PrPin Tg.S3 mice and transfected RK13 cells,but not N2a and SMB cells. These observa-tions, in conjunction with chelator studies,imply that b-processing of PrPC may occurby a mechanism distinct from metal cata-lyzed hydrolysis reported in vitro. In thiscurrent study, using mass spectroscopy anal-ysis of mouse brain S3 and Wt PrPC, wehave identified the “P1” amino acid residuefor b-cleavage in vivo. This information, aswell as studies with protease inhibitors, hasnarrowed the possible identities for a b-siteprotease. As alpha- versus beta-processing ofPrPC can lead to benign outcomes in thepresence of PrPSc or oligomeric A-Beta, ourfindings may provide useful insights into thepathogenesis of both prion and Alzheimer’sdisease.

P.32: A biochemical approach toevaluate the contribution to pathologicalprion protein formation from the 222KPrP variant in scrapie positive goats

Maria Mazza1, Chiara Guglielmetti1,Francesco Ingravalle2, Jan PM Langeveld3,

Loukia V Ekateriniadou4,Olivier Andr�eoletti5, and Pier Luigi Acutis1

1Genetics and Immunobiochemistry Laboratory;

Department of Neurosciences; Istituto

Zooprofilattico Sperimentale del Piemonte, Liguria

e Valle d’Aosta, Turin, Italy; 2Biostatistic;

Epidemiology and Risk Analysis Unit; Istituto

Zooprofilattico Sperimentale del Piemonte, Liguria

e Valle d’Aosta, Turin, Italy; 3Central Institute for

Animal Disease Control (CIDC-Lelystad), Lelystad,

The Netherlands; 4National Agricultural Research

Foundation; Veterinary Research Institute,

Thessaloniki, Greece; 5UMR INRA ENVT 1225;

Interactions HOtes Agents Pathog�enes;Ecole Nationale V�et�erinaire de Toulouse,

Toulouse, France

Lysine (K) at position 222 of goat PrP is asso-ciated with resistance to classical scrapie, yetfew natural cases of scrapie have been found inQ/K goats and intracerebral experimental trans-mission was successful in Q/K and K/K goatsafter long incubation periods. Previous WesternBlot (WB) studies performed by different mono-clonal antibodies (mAbs) revealed an inhibitionby K222 on the binding of mAb F99/97.6.1 togoat PrP. Thus a WB method, based on the ratiobetween the signal intensity given by mAbsF99/97.6.1 and SAF84, was developed to distin-guish goats exhibiting PrP encoded by the geno-types 222Q/Q, 222Q/K and 222K/K. Here weapplied this approach to investigate the contribu-tion to PrPSc formation given by the 222K vari-ant in scrapie positive goats. WB analyses wereperformed on the PrPC or PrPSC extracted fromthe brains of negative and scrapie positive goatsamples (natural or experimental), bearing dif-ferent genotypes at position 222. Samples wereanalyzed in triplicate and signals revealed byF99/97.6.1 and SAF84 were quantified. Adescriptive statistical analysis was performed.


No signals were detected by F99/97.6.1 inany of the 222K/K goat samples. The ratio ofthe optical densities revealed that the positive222Q/K goats had a similar reactivity to 222Q/Q samples. Halved values of the ratio werepresent in negative goats with 222Q/K. The sta-tistical analysis confirmed these differences.The obtained results showed that in heterozy-gous animals PrPSc seems to be formed nearlytotally by the Q222 variant thus confirming thehigher resistance to convertibility of K222 PrPvariant.

P.33: Genetic modulation of atypicalscrapie transmissions in transgenic

mouse models

Hae-Eun Kang1, Sehun Kim1,April Zander1, Thierry Baron2, and

Glenn Telling1

1Prion Research Center (PRC) and the Department

of Microbiology; Immunology and Pathology;

Colorado State University; Fort Collins, CO USA;2Agence Francaise de S�ecurit�e Sanitaire des

Aliments; Lyon, France

Atypical scrapie, a recently recognized andsurprisingly prevalent prion disease of sheep, isan example of an emerging prion disease ofuncertain origin and species tropism. Since itsdiscovery in 1998, precautionary testing haverevealed surprising numbers of atypical scrapiecases in Europe. In 2007, a sheep from a flockin Wyoming tested positive for atypical scrapie.Atypical and classical scrapie differ with bio-chemical features of the protease-resistant formof the prion protein (PrPSc), and neuropathol-ogy. Also, atypical scrapie occurs in sheep withPRNP genotypes usually associated with resis-tance to classical scrapie, and sheep with atypi-cal scrapie are more likely to expressphenylalanine (F) than leucine (L) at codon141. The origin and host range of atypical scra-pie are unclear. To address these issues, wegenerated transgenic mice expressing OvinePrP with phenylalanine at the residue 141, Tg(OvPrP-F141). We produced 2 sublines: thebrains of the Tg(OvPrP-F141)H express PrPC

»5-fold higher than wild type mice, while Tg(OvPrP-F141)L mice express at »2-fold. Tg(OvPrP-F141)H spontaneously developed trun-cal ataxia and limb paresis at a mean age of 70d. Remarkably, PrPSc accumulated in brainsand muscles of sick mice. Furthermore, weinoculated the Tg(OvPrP-F141)L, Tg(OvPrP-A136), and Tg(OvPrP-V136) mice with 6 atyp-ical scrapie isolates to determine the effects ofthe F141 genotype on atypical scrapie propaga-tion. Only Tg(OvPrP-F141)L developed clini-cal signs and the brain samples showed typicalbiochemical signature of atypical scrapie. Ourfindings suggest that atypical scrapie may havespontaneously originated and subsequentlypropagated and evolved by transmission insheep with susceptible genotypes.

P.34: Preliminary study of Alzheimer’sdisease transmission to bank vole

Guido Di Donato1, Geraldina Riccardi1,Claudia D’Agostino1, Flavio Torriani1,Maurizio Pocchiari2, Romolo Nonno1,

Umberto Agrimi1, andMichele Angelo Di Bari1

1Department of Food Safety and Veterinary Public

Health Istituto Superiore di Sanit�a, Rome, Italy;2Department of Cellular Biology and Neuroscience;

Istituto Superiore di Sanit�a, Rome, Italy

Extensive experimental findings indicatethat prion-like mechanisms underly the patho-genesis of Alzheimer disease (AD). Transgenicmice have been pivotal for investigating prion-like mechanisms in AD, still these models havenot been able so far to recapitulate the complexclinical-pathological features of AD. Here weaimed at investigating the potential of bankvole, a wild-type rodent highly susceptible toprions, in reproducing AD pathology uponexperimental inoculation.

Voles were intracerebrally inoculated withbrain homogenate from a familial AD patient.Animals were examined for the appearance ofneurological signs until the end of experiment(800 d post-inoculation, d.p.i.). Brains werestudied by immunohistochemistry for pTau


(with AT180 and PHF-1 antibodies) andb-amyloid (4G8).

Voles didn’t show an overt clinical signs,still most of them (11/16) were found pTaupositive when culled for intercurrent disease orat the end of experiment. Interestingly, volesculled as early as 125 d.p.i. already showedpTau aggregates. Deposition of pTau was simi-lar in all voles and was characterized by neuro-pil threads and coiled bodies in the alveus, andby rare neurofibrillary tangles in gray matter.

Conversely, b-amyloid deposition was ratherrare (2/16). Nonetheless, a single vole showedthe contemporaneous presence of pTau in thealveus and a few Ab plaque-like deposits in thesubiculum. Uninfected age-matched voles werenegative for pTau and Ab.

These findings corroborate and extend previ-ous evidences on the transmissibility of pTauand Ab aggregation. Furthermore, the observa-tion of a vole with contemporaneous propaga-tion of pTau and Ab is intriguing and deservesfurther studies.

P.35: Assessing milk from CWD-lactating deer for infectious prions

Erin McNulty, Amy Nalls,Jeanette Hayes-Klug, Kelly Anderson, and

Candace Mathiason


Transmissible spongiform encephalopa-thies (TSEs), or prions, cause a fatal neuro-degenerative disease in mammals includingbovine spongiform encephalopathy (BSE) incattle, scrapie in sheep, variant Creutzfeldt-Jakob disease in humans and chronic wastingdisease (CWD) in deer, elk and moose.CWD, the only prion disease to infect anative free-ranging population, has now beendetected in 23 US states, 2 Canadian provin-ces and South Korea. While horizontal trans-mission is credited for much of the spread ofCWD, few studies have monitored the poten-tial for vertical/maternal transmission withan emphasis on lactation. Using a small,polyestrous cervid—the Reeves’ muntjacdeer—we are addressing this issue by

supplementing na€ıve Reeve’s muntjac fawns(n D 5) with previously collected muntjacmilk. Each fawn (n D 3) was orally dosedwith pooled muntjac colostrum (6mls colos-trum at 24 hours) and milk (6mls milk perday for an additional 15 d [total of 90mls])from pre-clinical and clinical CWDC doe.Negative control fawns (n D 2) receivedsimilar colostrum/milk harvested from na€ıvedoe. CWD status of inoculated fawns andtheir dams will be monitored by immunohis-tochemistry, real time quaking induced con-version assay (RT-QuIC), protein misfoldingcyclic amplification (PMCA) and clinicaldisease progression. The results of this studywill establish: 1) if there are sufficient infec-tious prions in the milk of lactating doe totransmit disease to offspring and 2) if motherto offspring transmission plays a role in thehigh efficiency with which CWD is transmit-ted in nature.

P.36: Spontaneous in vitro conversion offull length recombinant human prion

protein in unseeded RT-QuIC reactions

Marcelo Barria Matus, Alexander Peden,Richard Knight, James Ironside, and

Mark Head

National CJD Research & Surveillance Unit; The

University of Edinburgh, Edinburgh, UK

Sporadic Creutzfeldt–Jakob disease (sCJD)is the most common human prion disease,affecting approximately 1–2 persons per onemillion of the population per year. It is thoughtto arise as a result of spontaneous conversionof PrPC to PrPSc, which becomes self-propagat-ing. The prion protein polymorphism at codon129 encodes either methionine (M) or valine(V). Comparison of the codon 129 genotypedistribution in sCJD cohorts with that of thenormal Caucasian population suggests that het-erozygosity (MV) protects against sCJD andthe comparison has also been widely inter-preted to mean that methionine homozygositypredisposes to CJD.

We have used real-time quaking inducedconversion (RT-QuIC) to model the


spontaneous formation of the abnormal form ofhuman PrP and to determine whether methio-nine or valine at the position 129 of PrPC con-fers a greater susceptibility to spontaneousconversion to PrP amyloid.

Unseeded RT-QuIC reactions using full-length recombinant human prion protein witheither methionine or valine at position 129 bothresulted in spontaneous amyloid formation.The process appeared to have a pronouncedstochastic element, but when a sufficient num-ber of replicates were performed a clear andreproducible effect of codon 129 genotype wasalso evident, in which PrPC with valine atcodon 129 showed a greater predisposition toform amyloid than its allelic counterpart con-taining methionine.

These results question whether methionine atposition 129 in PrPC can be considered anintrinsic susceptibility factor for conversion toPrPSc, at least in terms of the initiation of spon-taneous, as opposed to seeded PrP amyloidformation.

P.37: The differential distribution ofactin-cofilin rods in the brains of prion

inoculated transgenic mice

Bruce Pulford, Heather Bender, andMark Zabel

Colorado State University; Ft. Collins, CO USA

The neuropathology of transmissible spongi-form encephalopathies (TSEs) includes the lossof synapses and neurons within the central ner-vous system (CNS). Potential causes of thisCNS pathology include glutamate toxicity andoxidative stress, which have also been shownto induce ADF/cofilin rods within neuronal pro-cesses in both cultured hippocampal neuronsand Alzheimer’s disease brain. Cofilin rods cancompletely occlude neurites leading to loss offunction distal to the rods. In this study, we areinterested in determining if there are increasesin the presence of ADF/cofilin rods in prion-inoculated transgenic mouse brains leading tothe loss of neuronal processes and ultimatelyneurons as the disease progresses. Tg5037

mice, expressing cervid PrP (prion protein),and TgA20 mice, overexpressing mouse PrP,were ic inoculated with E2 (CWD elk brainhomogenate) and RML (Rocky Mountain Labsmouse adapted scrapie) respectively. Controlsincluded Tg5037 and TgA20 mice inoculatedwith normal brain homogenate (NBH) andFVB and PrP% mice inoculated with E2 orRML. TgA20 mice were euthanized at 40dpi,50dpi, and when clinical signs appeared;Tg5037 mice were euthanized at 80dpi, 120dpi,and when terminal. Brains were removed,fixed, frozen or wax embedded, sectioned andexamined by immunostaining for the presenceof cofilin rods and PrPSc. In Tg5037 mice, rodsoccurred predominantly in the inferior olivarycomplex in the brainstem. In TgA20 mice, rodsbegan to appear at 40dpi in the superficial cor-tex, and spread to deeper areas of the cortexand other regions of the brain by 50dpi.

P.38: Deletion of the C-terminal part ofprion protein (PrPC) leads to ER

retention, p38 MAPK activation andneurodegeneration in transgenic mice

Berta Puig1, Hermann Altmeppen1,Sarah Ulbrich2, Susanne Krasemann1,Karima Chakroun1, J€org Tatzelt2, and

Markus Glatzel1

1Institute of Neuropathology; University Medical

Center Hamburg-Eppendorf, Hamburg, Germany;2Institute of Biochemistry und Pathobiochemistry;

Ruhr University, Bochum, Germany

Many aspects of PrPC biology and conver-sion to PrPSc are still enigmatic. Transgenicmice lacking PrPC (Prnp0/0) do not display anygross disease phenotype that could shed lightinto the mechanisms of neurodegeneration.However, some transgenic mice with deletionsat the N-terminal and central domains or inser-tions and point mutations in PrPC, present aclinical phenotype reminiscent of prion disease.Interestingly, several mutations associated withhuman prion diseases are clustered in the C-ter-minal part of PrPC, which probably affectsPrPC stability and leads to the misfolding of its


structured globular part. Point mutationslocated here also show transmission propertiesin transgenic mice.

We have studied a deletion mutant of PrPC

lacking 16 C-terminal amino acids (PrPD214–229) between the disulfide bond (from Cys178to Cys213) and the omega site for GPI-anchorattachment (Ser230). In cells, we observed thatthis deletion interferes with the maturationthrough the secretory pathway as PrPD214–229and it is retained in the ER. Remarkably, miceexpressing PrPD214–229 present with a neuro-logical disease showing a different clinical pre-sentation compared to classical prion disease.In these mice, PrPD214–229 is also partiallyretained in the ER and PK resistant. Impor-tantly, we show a clear activation of p38MAPK both at pre-clinical and clinical timepoints. To our knowledge, this is the first timethat aberrant trafficking of PrP is associatedwith a specific signaling pathway leading toneurodegeneration and a neurological diseasein mice.

P.39: Essential collective dynamicsanalysis of unfolded a-synuclein dimers

Jonathan Mane1,2 and Maria Stepanova1,2

1Department of Electrical and Computer

Engineering; University of Alberta, Edmonton,

Alberta, Canada; 2National Institute for

Nanotechnology; National Research Council

Canada, Edmonton, Alberta, Canada

The collective motions of group of atoms ina protein influence its folding and stability, andmay help identify early stages of protein mis-folding. In this study, we looked into the fold-ing dynamics of a-synuclein protein dimers.a-Synuclein is localized in the presynapticnerve terminals found mainly in the brain tis-sue. Its misfolding is linked to certain neurolog-ical disorders like Parkinson disease. Weinvestigated the folding of a-synuclein via all-atom molecular dynamics (MD) simulationsstarting with unfolded dimer models withoutany secondary structures. The resulting MDtrajectories were analyzed using both

conventional analysis of secondary structuresand essential collective dynamics (ECD) - anovel methodology for identification ofdynamic structural domains of correlatedmotions; analysis of the local flexibility of pro-teins; and construction of correlation maps thatuncovers the existence of networks of dynami-cally correlated groups of atoms. In the courseof the MD simulations, we found that the ini-tially unfolded a-synuclein dimers collapse butretain most of their local alignment. After about5–10 ns, they developed pronounced parallel oranti-parallel b-sheets. The arising b-sheetsoften belong to single ECD dynamical domainsindicative of relatively rigid structures movingcoherently. When b-sheets are formed involv-ing 2 adjacent chains of the dimer, the ECDflexibilty profiles of the main-chain atoms tendto show similar levels of flexibility for bothchains confirming a build-up of rather uniformdynamics in these regions. Pair correlationmaps suggest that immediate interatomic con-tacts, rather than distant interactions, appear todetermine the dynamics trends in the dimers.

P.40: Prion strain interference is acommon property of prions

Katie Langenfeld and Jason Bartz

Creighton University, Omaha, NE USA

Prion strain interference involves one strainblocking the emergence of a second strain andmay be involved in prion adaptation. The longincubation period drowsy (DY) strain of trans-missible mink encephalopathy (TME) caninterfere with several short incubation periodstrains by many different routes of infection. Itis unknown if other long incubation periodstrains can interfere with short incubationperiod strains. To explore this, we utilized thelong incubation period 139H strain of hamster-adapted scrapie as the blocking strain. Sciaticnerve inoculation of 139H 25, 50 or 75 d priorto superinfection with the short incubationperiod hyper (HY) TME strain resulted in the25-day and 50-day interval groups having incu-bation periods and clinical signs similar to HY


TME. Conversely, 4 of the 5 animals in the 75-day interval group had 139H clinical signs andincubation period. Clinical diagnosis was con-firmed based on the strain-specific PrPSc con-formational stability profiles. Based upon theseresults, 139H was able to block the emergenceof HY TME in the 75-day interval group, indi-cating that strain interference had occurred. Wethen determined that sciatic nerve inoculationof 139H results in transport of PrPSc along thesame 4 descending motor tracks as HY and DYTME indicating that 139H strain interferenceoccurs in ventral motor neurons of the lumbarspinal cord. Overall, these studies indicate thatprion strain interference is a common propertyof prions and has implications for the mainte-nance and evolution of prion strains.

P.41: Sporadic distribution of prion-forming ability of Sup35p and Ure2p

from yeasts and fungi

Herman Edskes and Reed Wickner

National Institutes of Health; NIDDK, Bethesda,

MD USA

The Ure2p and Sup35p are 2 of several pro-teins capable of forming infectious amyloid inthe yeast Saccharomyces cerevisiae. Ure2pfunctions in the ability of the cell to respond tothe quantity and quality of nitrogen sourcesavailable in the growth environment and canform the [URE3] prion. Sup35p is part of thetranslation termination machinery and can formthe [PSIC] prion. For both proteins their func-tionality is compromised upon prion formation.Both proteins also show remarkable evolution-ary conservation of their domain structures.The domains of Ure2p and Sup35p that formamyloid are devoid of defined secondary struc-ture. Nevertheless these domains are function-ally important: binding microtubules andcontrolling mRNA turnover in Sup35p and sta-bilizing Ure2p. Homologues of Sup35p andUre2p from other yeasts have been shown to becapable of prion formation implying that prion-forming ability of these proteins is conservedin evolution and thus has survival/fitness value

for these organisms. The presence of eitherprion can have a substantial negative effect onthe cells, especially de novo generated prions.Even the mildest variants of these prions arerare in wild isolates. We surveyed a number ofyeast and fungal species for the ability of theirSup35 or Ure2p homologues to form prions inS. cerevisae. We find that many of the assayedproteins could not form prions suggesting thatprion-forming ability is not a conserved traitbut is a side effect of a domain conserved foranother function.

P.42: PrPSc-containing brain samplesinduce PrPC-dependent retraction ofdendritic spines in primary neurons

Cheng Fang and David Harris

Boston University, Boston, MA USA

Despite intensive investigation, how PrPSc

induces neuronal degeneration is still unknown.Synaptic loss is an early event in prion disease,based on immunostaining of infected brain sli-ces (Mallucci et al. Neuron 53, pp325–3352007). However, the cellular and molecularmechanisms underlying this process have beendifficult to study, primarily because of theabsence of systems for modeling PrPSc-inducedsynaptotoxicity in vitro. In our lab, we havebeen attempting to develop such a model sys-tem. Thus far, we have now shown that PrPSc-containing brain homogenates from infectedmice induce dendritic spine retraction and syn-aptic loss in mature mouse hippocampal neu-rons. In contrast, brain homogenate fromnormal brain does not induce synaptic loss.Further, we observed that the effect is PrPC-dependent, as PrPSc-containing brain homoge-nate does not induce synaptic loss in neuronsfrom PrP knockout mice. Interestingly, the syn-aptotoxic effect requires the N-terminal domainof PrPC. When neurons cultured from trans-genic mice expressing an N-terminally deletedform of PrPC (D23–111) were treated withinfected brain homogenate, no synaptic losswas observed. These results establish a cell


culture system for analyzing how PrPSc affectsneuronal function and synaptic integrity.

P.43: Cell culture models for studyingCWD prions

Shubha Jain, Sampson Law,Yuzuru Taguchi, Jeff Biernaskie, and

Hermann M Schatzl

Department of Comparative Biology and

Experimental Medicine; Faculty of Veterinary

Medicine; University of Calgary; Calgary,

AB, Canada

Chronic wasting disease (CWD) of cervids isthe most contagious prion disease. CWD prioninfectivity is found in many tissues, body fluidsand secretions. Cell culture models provide theopportunity to study the cellular and molecularbiology of prion infections. Mostly scrapie-derived and mouse-adapted prions have beenstudied in cell models. Currently, only modifiedRK13 cells are available as persistentlyinfected model for CWD prions. We want todevelop new cell culture models for acute andpersistent propagation of CWD prions. Ourrational is to use cell lines derived from cervidtissue which endogenously express cervid PrPc.We have established skin-derived stem cellsfrom antler tissue of Caribou as such stem cellsare multipotent and allow differentiation intoother lineages. Importantly, prion infection ofantler velvet was found in vivo. In addition, weuse a non-transformed fibroblast cell line fromIndian Muntjac deer, as fibroblasts are knownto be well susceptible to prion infection. Usingimmunoblot and confocal microscopy analysiswe found that both cell lines express normallyglycosylated PrPc and that PrPc is located at theouter leaflet of the plasma membrane. We arenow in the process of infecting these cells withCWD prions derived from brains of transgenicmice or deer and elk. PrPSc uptake and prionpropagation is studied using immunoblot andconfocal microscopy read-outs. Novel CWDprion propagating cells will be useful for eluci-dating the molecular mechanisms underlyingthe pronounced lateral spread of CWD prions.

P.44: The influence of proteinase-activated receptor 2 deficiency on the

course of prion infection: Anexperimental study using murine model

Zdenka Hanusova1, Tibor Mosko1,Zuzana Jindrova1, Radoslav Matej2, and

Karel Holada1

1Institute of Immunology and Microbiology; First

Faculty of Medicine; Charles University in Prague;

Prague, Czech Republic; 2Department of Pathology

and Molecular Medicine; National Reference

Laboratory for Human Prion Diseases; Thomayer

Hospital; Prague, Czech Republic

Background. Proteinase-activated receptors(PARs) play an important role in modulationof many pathological processes includingneurodegeneration. Recently, we have dem-onstrated that deletion of trypsin receptorPAR-2 prolongs survival of mice intracere-brally inoculated with RML prions.1 PAR-2is expressed in many cells and tissuesincluding immune organs which are involvedin peripheral prion propagation. The aim ofour study is to evaluate the effect of PAR-2deletion on survival of mice after peripheralinoculation with prions.

Methods. PAR-2C/¡ heterozygous mice werebred to obtain littermates sharing the samegenetic background except for PAR-2 expres-sion (PAR-2¡/¡, PAR-2C/¡, PAR-2C/C). 48sibling mice were inoculated subcutaneouslywith high dose of RML prions. To prevent theinfluence of subjective factors, the experimentis blind and mice were not genotyped. Mice areobserved for signs of scrapie and their weightis monitored. Mice in terminal stage of the dis-ease are euthanized and organs are harvestedfor genotyping and further analyses.

Results. To date, 27 out of 48 infected micewere euthanized. Mean survival and number ofanimals in the genotype groups are as follows:PAR-2C/C (232.5 § 6.8 d, n D 6); PAR-2C/¡

(232.6 § 9.8 d, n D 16); PAR-2¡/¡ (237.0 §7.6 d, n D 5). All remaining infected mice aresymptomatic.


Conclusions. Our preliminary data suggestthat the survival of mice after peripheral inocu-lation with prions does not seem to be notablyaffected by PAR-2 deletion. Final data will bepresented at the meeting.

Funding. The study was supported by proj-ects: GACR-P303/12/1791, GAUK-1322713,IGA-NT14145–3, PRVOUK-P24/LF1/3.

Reference

1 Matej R, et al. J Gen Virol 2012; 93:2057–61;

PMID:22694901; http://dx.doi.org/10.1099/vir.0.

043877-0

P.45: Peptide aptamers binding to PrPCinhibit prion protein misfolding

Erica Corda1, Xiaotang Du2,Jessica Siltberg-Liberles3, and

Sabine Gilch1

1Department of Ecosystem and Public Health;

Faculty of Veterinary Medicine, University of

Calgary; Calgary, Alberta, Canada; 2University of

Wyoming; Laramie,WY USA; 3Florida International

University; Miami, FL USA

Currently neither therapeutic nor prophylac-tic tools are available for treatment of prion dis-eases. In order to identify anti-prioncompounds, we aim to inhibit prion conversionby interfering with the interaction between thecellular prion protein PrPC and its infectiousform PrPSc, which represents the first step inthe pathogenesis of these fatal disorders.Wehave previously described a peptide aptamer(PA8) binding to PrPC which reduces prionpropagation in prion infected cultured cells.Peptide aptamers consist of combinatorial pep-tides inserted into a scaffold protein. Toimprove binding affinity, and thereby the anti-prion activity of PA8, its interaction with PrPChas been modeled in silico. Three residues ofthe 16mer peptide were defined as targets foramino acid substitutions that are expected tostrengthen the PA8-PrPC interaction. Accord-ing to these results, we have created selectedPA8 variants by introducing single amino acid

exchanges in the peptide moiety. All PA8 var-iants were expressed in E. coli, purified viaHis6-Tag and employed for treatment of prion-infected neuroblastoma cells. Using differentconcentrations of purified peptide aptamers, weidentified three variants that have shown anenhanced inhibition of PrPSc conversion incomparison to the original PA8. We plan toevaluate their activity also by transfection ofpersistently scrapie infected cells with PA8 var-iants that harbor cellular targeting signals andin mouse bioassays.In future, our results will beuseful for identifying a compound by rationaldrug design based on the structure of the PA-PrPC complex which is able to counteract prionconversion in vivo.

P.46: Mitigation of prion infectivity andconversion capacity by a simulatednatural process—repeated cycles of

drying and wetting

Qi Yuan2,*, Thomas Eckland2,Glenn Telling3, Jason Bartz2, and

Shannon Bartelt-Hunt1

1University of Nebraska-Lincoln; Omaha, NE, USA;2Creighton University; Omaha, NE, USA;

3Colorado State University; Fort Collins, CO, USA

Prions enter the environment frominfected hosts, bind to a wide range of soiland soil minerals, and remain highly infec-tious. Environmental sources of prionsalmost certainly contribute to the transmis-sion of chronic wasting disease in cervidsand scrapie in sheep and goats. While muchis known about the introduction of prionsinto the environment and their interactionwith soil, relatively little is known aboutprion degradation and inactivation by naturalenvironmental processes. In this study, weexamined the effect of repeated cycles ofdrying and wetting on prion fitness anddetermined that 10 cycles of repeated dryingand wetting could reduce PrPSc abundance,PMCA amplification efficiency and extendthe incubation period of disease. Importantly,prions bound to soil were more susceptible


to inactivation by repeated cycles of dryingand wetting compared to unbound prions, aresult which may be due to conformationalchanges in soil-bound PrPSc or consolidationof the bonding between PrPSc and soil. Thisnovel finding demonstrates that naturally-occurring environmental process can degradeprions.

P.47: An apparently universal substratefor RT-QuIC-based detection anddiscrimination of prion strains

Christina D Orru�1, Bradley R Groveman1,Lynne D Raymond1, Andrew G Hughson1,

Romolo Nonno2, Wenquan Zou3,Bernardino Ghetti4, Pierluigi Gambetti3,

and Byron Caughey1


Mountain Laboratories; NIAID; NIH; Hamilton,

MT, USA; 2Department of Veterinary Public Health

and Food Safety; Istituto Superiore di Sanit�a;Rome, Italy; 3Department of Pathology and

Laboratory Medicine; Indiana University School of

Medicine; Indianapolis, IN, USA; 4Department of

Pathology; Case Western Reserve University;

Cleveland, OH USA

Prions propagate as multiple strains and canbe found in an assortment of sub-types in manymammalian species. The detection of all suchprion populations by a single ultrasensitiveassay would be valuable in prion disease diag-nosis, surveillance and research. Here we showthat under novel conditions a previouslyuntested recombinant prion protein is an effec-tive substrate for the sensitive RT-QuIC detec-tion of a total of 26 prion types/strains testedthus far from humans, cattle, sheep, cervids androdents. These prion strains include several thatwere previously undetectable, such as sheepNor98 scrapie and some human GSS prionseeds. Furthermore the ability of different prionseeds to induce polymerization of specific RT-QuIC substrates can be exploited to discrimi-nate prion strains such as sporadic and variantCreutzfeldt-Jakob disease in humans, classicaland atypical Nor98 scrapie strains in sheep, and

classical and atypical BSE in cattle. Moreover,striking strain-dependent differences in the pro-tease-resistant banding profiles of the RT-QuICconversion products was observed, suggestingthe existence of multiple distinct classes ofprion templates and additional means of straindiscrimination.

P.48: Biochemical properties of chronicwasting disease prions and relationship

with prion shedding

Samia Hannaoui1, Barbara Killy1,Stefanie Czub2, Valerie Sim3, and

Sabine Gilch1

1Department of Ecosystem and Public Health;

Faculty of Veterinary Medicine; University of

Calgary; Calgary, Canada; 2Canadian Food

Inspection Agency Lethbridge Laboratories;

Lethbridge, Canada; 3Centre for Prions and Protein

Folding Diseases; University of Alberta; Edmonton,

Canada

Chronic wasting disease (CWD) is the mostcontagious prion disease with substantial lateraltransmission. The appearance of CWD in wild-living and migrating cervids makes it uncon-trollable. Therefore, potential risk for humanand other animals still remains. In most priondiseases, abnormal prion protein (PrPSc) ismainly found in the brain. However, in CWD(alike scrapie), PrPSc is detectable in manyextraneural tissues, which contributes to therapid spread of the disease. Studies suggest alink between biochemical stability of strains,incubation time of disease and neuroinvasiveproperties. These data led to our hypothesisthat prion transport and shedding depend onintrinsic biochemical properties of strains.Thus, we analyze the PrPSc aggregate composi-tion, proteinase K (PK) resistance and confor-mational stability of CWD-elk, -deer, -white-tailed deer and -mule deer, and ME7 scrapieprions. Our results suggest similar PK-resis-tance of CWD and scrapie strains. However,the conformational stability (GdnHCl1/2) ofthese prion strains is different. Moreover,velocity sedimentation fractions of CWD and


scarpie strains show a different distribution.PrPSc from CWD-elk is found in fractions witha lower density. These results suggest a differ-ent aggregate-stability and -size distribution.We aim to elucidate whether CWD prions havecertain properties that make them more proneto be transported from the brain to peripheralorgans. Our results will add novel knowledgeabout pathogenesis of CWD and might eluci-date targets that help to prevent prion sheddingwhich may help further spread of CWD andthus, reduces a potential future risk to humanand animals (cervids and non-cervids).

P.49: 2-cysteine-substitution mutants ofPrP for studying the interfaces of PrPC-

PrPSc interaction

Yuzuru Taguchi and Hermann Sch€atzl

University of Calgary; Calgary, AB Canada

We introduced 2-cysteine-substitutions (C;C-PrP) into PrP, one at a residue in the regionbetween helix 1 and 2 (H1»H2) and the otherone between residues 220 and 229 (Ctrm), toform an additional disulfide bond and to intro-duce structural constraints into PrPc. Interest-ingly, depending on positions of the cysteine inCtrm, such PrPs were converted into PrPSc inpersistently prion-infected cells. We found thatC;C-PrPs with 165C-229C, 166C-229C, and168C-225C showed substantial levels of PK-resistant fragments. These findings implied thatthe H1»H2 portion can undergo a positionalchange toward Ctrm during interaction withPrPSc. Next, we assessed influences of disulfidecrosslinks on dominant-negative inhibition(DNI). We introduced a deletion of residue 159(D159) to C;C-PrP constructs (DC;C-PrP) andobserved their DNI in 22L-infected N2a cells.DNIs of DC;C-PrPs were highly affected bypositioning of the cysteine in Ctrm: some C;C-PrPs substantially lost DNI by combinationwith D159, while others exerted efficient DNI.These results imply that the aforementionedpositional change of H1»H2 and the subcellu-lar localization of PrPc constructs influencehow PrPC interacts with PrPSc. It also suggests

that, when affinity of one interface is weak,another interface might take over as a maininterface for PrPC-PrPSc interaction. Such ashift of main interface might occur during het-erologous PrPC-PrPSc interaction in interspe-cies transmission or depending onmicroenvironments and cofactors. Thus, studieswith C;C-PrPs will help to elucidate the molec-ular and cellular mechanisms in the interactionof PrPC with PrPSc which is a prerequisite inprion conversion.

P.50: Assessment of high pressuredigestion prion mitigation by bioassay

Laura Pulscher, Erin McNulty, Amy Nalls,Craig Ramsey, and Candace Mathiason


Approximately 150 million people andalmost $40 billion worth of agricultural com-modities pass through US international portsannually. Ports seize animal and plant productspotentially contaminated with high risk dis-eases that then must be decontaminated. In thisstudy we assess the efficacy of alkaline diges-tion to mitigate infectious prions. Transmissiblespongiform encephalopathies (TSEs), or prions,were chosen as the infectious agent for thisstudy because they are difficult to inactivate,affect both human and animal species world-wide and are shed by infected hosts into theenvironment establishing highly infectiousbiota. Chronic wasting disease (CWD), theTSE of cervid species in North America, hasrecently been spotlighted as a potential concernfor European countries, and recapitulateshuman and animal TSE pathogenesis. We proc-essed CWD positive and negative brain tissueby alkaline digestion under standard tempera-ture and pressure at time intervals of 2, 4, and6 hours. Digested tissues were analyzed forresidual infectivity by bioassay into transgenicmice expressing the cervid prion protein(CerTg5037) and amplification competentprions by in vitro RT-QuIC methodology.While bioassays are ongoing (330 dpi), prelim-inary results indicate that prion contaminated


tissues may be rendered noninfectious after asingle 4 hour cycle of alkaline digestion. Thiswork will provide a basis for future studiesdesigned to unravel mechanisms associatedwith the ability of prions to bind surfaces,enhancing prion mitigation strategies.

P.51: Targeted zebrafish mutants tounveil normal functions of, and

interactions between, prion protein andamyloid precursor protein: Relevance to

Alzheimer disease

Patricia LA Leighton and W Ted Allison

University of Alberta; Edmonton, AB Canada

Objective. We aim to uncover the elusivefunctions of prion protein (PrP) and amyloidprecursor protein (APP) and to identify thera-peutic targets for prion diseases and Alzheimerdisease. Zebrafish have 2 homologs of PRNP(prp1 and prp2) and APP (appa and appb) thathave functional conservation with their mam-malian counterparts. Our demonstration of agenetic interaction between prp1 and appa sup-ports novel roles for PrP in Alzheimer disease(Kaiser et al. 2012 PMID: 23236467).

Methods. We recently created and character-ized zebrafish with a null prp2 allele (Fleischet al. 2013 PMID: 23523635). Here we mea-sured c-fos expression by in situ hybridizationand qPCR as a molecular indicator of neuronactivity in zebrafish exposed to the convulsantpentylenetetrazole (PTZ). We used TAL Effec-tor Nucleases (TALENs) to mutagenize zebra-fish prp1 and appa.

Results. PTZ treatment induced significantlymore c-fos expression in 2 dpf prp2¡/¡ larvaerelative to wild type. We also engineered novelzebrafish with TALEN-induced frameshiftmutations in appa and prp1. Assessing pheno-types in zygotic prp1¡/¡, appa¡/¡ and com-pound mutants is underway.

Discussion. Enhanced expression of c-fos in 2dpf prp2¡/¡ larvae compared to wild type

larvae after PTZ exposure further supports aprotective role for PrP2 against neuronal hyper-excitability. This phenotype provides a plat-form to map neuroprotective functions to spe-cific PrP protein domains via mRNA rescueexperiments. Our novel Prp1 and appa mutantswill be bred to study interactions between PrPand APP paralogs. Our findings have importantimplications for design of prion disease andAlzheimer disease therapeutics.

P.52: Pentosan polysulfate initiates prionfibrillization by inducing intramolecular,

hydrophobic interactions of theoctarepeat with the C-terminus

Martin Windsor, Hernan Carol, andDavid Cullis-Hill

Sylvan Scientific; Sydney, NSW, Australia

We used thioflavin (ThT) fluorescence,intrinsic tryptophanyl fluorescence (ITF) andcircular dichroism (CD) spectroscopy to mea-sure hydrophobic transitions in the solventenvironment of the PrP octarepeat (OR) trypto-phanyl side chain cluster (ORtc) during fibrilformation induced by the synthetic heparinoidpentosanpolysulfate (PPS). By studying spec-tral intensity and wavelength shifts in fulllength prion protein (PrP23-231), N-terminallytruncated material (PrP112-231, PrP90-231) andPrP octarepeat peptide (OR, PrP53-90) weobserved that the ORtc is in a more hydrophobicenvironment in native PrP23-231 than in free ORpeptide (PrP53-90). This indicates an intermo-lecular hydrophobic burial of the ORtc in PrP23-231. PPS induced rapid fibril formation and anincrease in ITF of PrP23-231 and PrP90-231. Fibrilformation by PrP90-231indicates there is a C-ter-minal PPS-binding site.

We conclude that in PrP23-231, the N-termi-nal ORtc has intramolecular affinity for the C-terminal hydrophobic domain (HD, PrP112-122)and when bound to PPS forms a tertiary, hydro-phobic complex, which leads to fibrilization.We propose that 1) heparinoid molecules mayinduce a PrP oligomerization to a dodecamerwhich is biologically active; 2) a defective


heparan structure may result in induction of astable pathological PrP fibril structure.

P.53: Active vaccination usingmultimeric PrP as immunogen asstrategy to contain chronic wasting

disease

Dalia Abdelaziz, Shikha Jain,Sandi Nishikawa, Sampson Law,

Angelo Bianchi, and Hermann Schatzl

University of Calgary; Calgary, AB, Canada

Chronic wasting disease (CWD) is the onlyprion disease which occurs in free ranging andcaptive animals. It expands geographically andin numbers in North America and the potentialfor transmission into humans cannot beexcluded. Effective measures for containingCWD are therefore crucial. It is our centralhypothesis here that it is feasible to interfere inperipheral prion infection and prion sheddingby inducing auto-antibodies against PrPc byactive vaccination. Our rationale is to over-come self-tolerance against PrP by using ab-sheeted multimeric recombinant PrP asimmunogen. As our previous experimental datain wild-type mice have shown, this approachinduces robust humoral and cellular responsesagainst PrP, notably without adverse sideeffects. Auto-antibodies neutralized prion prop-agation in cultured cells and up to 25% ofimmunized and prion challenged mice survivedprion infection. Having a reliable proof-of-con-cept for our strategy in wild-type mice we arenow focusing on active vaccination againstCWD. We expressed in E.coli, purified andrefolded 4 immunogens: cervid and murine PrPin monomeric and dimeric form. As deliverystrategy allowing oral vaccination we co-encapsulated recombinant PrPs with variousadjuvants into microspheres. For testing immu-nogenicity and safety we are currently perform-ing immunization studies in wild- type andcervid PrP-transgenic mice. Protection againstCWD will be tested by infecting immunizedmice with mouse and cervid prions. Our long-term goal is to implement a translational

research program for developing a wild-lifesuitable vaccine which is able to reduce levelsof morbidity and mortality and further trans-mission of CWD.

P.54: Infectious recombinant prions: Invitro generation and propagation of

different strains

N Ferna�ndez-Borges1, MA Di Bari3,H Era~na1, E Vidal5, V Venegas1,

AM Sevillano6, SR Elezgarai1, L Pirisinu3,E V�azquez-Fern�andez7, C Harrathi1,

B Parra8, C D’Agostino3, JC Espinosa9,W Surewicz10, JM Torres9, T Mayoral8,U Agrimi3, JR Requena6, R Nonno3, and

J Castilla1,2

1CIC bioGUNE; Derio, Spain; 2IKERBASQUE;

Bilbao, Spain; 3Istituto Superiore di Sanit�a; Rome,Italy; 4University Salamanca; Salamanca, Spain;

5Priocat-CReSA; Bellaterra, Spain; 6CIMUS-USC;

Santiago de Compostela, Spain; 7University of

Alberta; Edmonton, Canada; 8Laboratorio Central

de Veterinaria; Madrid, Spain; 9CISA-INIA;

Madrid, Spain; 10CEAFA; Cleveland, USA

Prion diseases are a group of fatal neurode-generative diseases that affect humans and ani-mals and whose main characteristic is itsinfectious nature. PrPSc, a misfolded variant ofthe endogenous PrPC, is the solely pathogenicagent. The infectivity of the misfolded proteinwas amplified/propagated in vitro since adecade ago using Protein Misfolding CyclicAmplification (PMCA), a technology that hashad an enormous impact in the prion field.Recently, a version of PMCA using recombi-nant PrP (rec-PrP) as substrate (rec-PMCA) hasbeen developed to generate highly PK resistantPrP (rec-PrPres). The infectivity shown by adiversity of rec-PrPres generated in vitro bydifferent groups using a variety of co-factorsand modified procedures was also diverse.These results confirm: (i) the GPI and glycosyl-ation components are not necessary in enci-phering an infectious conformation and (ii) rec-PrPres can be also structured in the form of


different recombinant prion strains with robustin vitro self-replicating abilities but dissimilarinfectious features in vivo.

Our study has been focused on understand-ing the infectivity and the effect of differentcofactors of recombinant prions generatedusing the polymorphic variant of the bank volePrP (109I). This model was used as the shortestincubation time model for prion diseases andbecause of its outstanding susceptibility topropagate most of the existing prion strainsfrom different species.

This study shows the in vitro generation ofinfectious recombinant bank vole prions and howcofactors influence the propagation of certainprion strains with specific infectious features.

P.55: Discrimination of classical andatypical BSE by a distinct PrPSc profile

Christine Fast1, Catherine Graham2,Martin Kaatz3, Kristina Santiago-Mateo3,

Tammy Pickles2, Kendra Sullivan3,Anne Balkema-Buschmann1,

Martin H Groschup1, and Stefanie Czub2,3

1Friedrich-Loeffler-Institut; Isle of Riems,

Germany; 2Canadian Food Inspection Agency;

Lethbridge, Alberta, Canada; 3University of

Calgary; Veterinary School; Department of

Production Animal Health; Calgary,

Alberta, Canada

Bovine spongiform encephalopathy (BSE)belongs to the transmissible spongiform enceph-alopathies and is associated with the accumula-tion of a pathological isoform of a host-encodedglycoprotein, prion protein (PrPSc). To date,classical BSE (C-type) and 2 atypical BSE forms(L- and H-type), are known and their discrimina-tion is based on the biochemical characteristics.The challenge to surveillance programs is to dif-ferentiate classical BSE from atypical BSEcases, since C-type BSE is feed borne and atypi-cal BSE still of unknown origin. However, onlyWestern blot analysis allows a clear discrimina-tion between the BSE forms until now. The goalof our study was to identify type-specific PrPSc

profiles by using Immunohistochemistry as anadditional method.

In total 21 cattle, intracerebrally inoculatedwith C-type, H-type and L-type BSE wereused. From these animals, 6 well-defined brainareas were examined by H&E staining and byImmunohistochemistry (IHC), using 2 differentC-terminal antibodies.

The histopathological examination revealedclear differences among the individual cattle,but the immunohistochemical data pointstoward the existence of a distinct PrPSc profilebetween the different BSE-types. This profileinvolved both the specific brain areas affectedand the cellular pattern of the PrPSc deposition.

The qualitative and quantitative analysis ofPrPSc affected structures shed new light intothe pathogenesis of the different BSE types.Additionally, the results presented here supportIHC characterization as additional diagnostictools in classical and atypical BSE surveillanceprograms, in particular when only formalinfixed tissue samples are available.

P.56: Factors that improve RT-QuICdetection of prion seeding activity

Andrew Hughson, Christina Orru,Bradley Groveman, Katrina Campbell, and

Byron Caughey

Rocky Mountain Labs; NIH/NIAID; Hamilton,

Montana USA

The Real-Time Quaking-Induced Conversion(RT-QuIC) assay for prion-associated seedingactivity has been applied successfully to awide variety of transmissible spongiformencephalopathies (TSEs) and tissues and isbeing implemented in ante mortem diagnostictesting. Our laboratory has continued to exploreassay factors in order to assess and improve per-formance and practicality. Included in the manyreaction parameters that we have evaluated aresubstrate selection, temperature, shaking condi-tions, pH and NaCl concentrations. Our resultsindicate that a high degree of sensitivity andspecificity are achieved by optimizing these con-ditions within certain ranges. We demonstrate


that replacing full length recombinant prion pro-tein (rPrPSen) substrate with N-terminally trun-cated rPrPSen (residues 90–231) decreases theassay’s lag time. Similar effects were observed,without compromising specificity, when eitherthe reaction temperature or the shaking speedwas increased. An optimization of concentra-tions between 130mM and 300mM NaCl pro-vides specificity by reducing prion-independentamyloid formation. Furthermore, we find thatpH 7.4 provides for more rapid and prion-spe-cific RT-QuIC reactions compared to moreacidic pH’s. When a cumulative optimization ofthese conditions was applied to nasal brushingsfrom human sporadic Creutzfeldt-Jakob diseasepatients, higher temperatures reduced RT-QuIClag phases, and the use of hamster recombinantprion protein 90–231 as substrate strengthenedprion-associated signals. Collectively, theseresults demonstrate improved speed, efficacyand practicality of RT-QuIC assays and high-light variables to be optimized for applicationsto new prion strains and sample types.

P.57: Examining the role of PrPc inregulating neuronal activity and

myelination

D Ezekiel Watson, Arsalan S Ahmed,Declan W Ali, and W Ted Allison

University of Alberta; Edmonton, Canada

Our research uses zebrafish to examine thefunction of PrPc in an intact in vivo animalmodel. Previously our lab has shown alteredNMDA receptor kinetics in the Mauthner cellof PrP2¡/¡ larvae (Fleisch et al. 2013PMID:23523635). We are developing a simpleand reliable behavioral assay for evaluating therole of PrPc in regulating synaptic activity byexploiting the Mauthner cell controlled touchevoked escape response (TEER) in larvalzebrafish. Thus far, results show that PrP2¡/¡

larvae take 55% longer to complete thisresponse (p D 0.002). This assay, combinedwith microinjection of domain swapping PrPc

mRNA constructs, will allow us to dissect therelevant domains of PrPc for regulating

neuronal excitability. Altered axonal conduc-tance, a phenotype observed in PrP¡/¡ miceexhibiting demyelination in their PNS, maycontribute to this longer duration TEER. Con-sequently, we have evaluated the condition ofmyelin in the posterior lateral line nerve ofPrP2¡/¡ zebrafish, a PNS nerve important fortactile sensation. Preliminarily, in aged PrP2¡/

¡ zebrafish, we do not observe a similar thin-ning of the myelin as was seen in mice, butmyelin and axons do display significantlyaltered structure. To better examine the func-tion of PrP2 in myelin maintenance we havebeen testing the ability of the antibody SAF84to detect zebrafish PrP2. Thus far our testingsuggests that SAF84 is indeed capable ofdetecting zebrafish PrP2. In summary, the datademonstrate that PrPc knockout zebrafish aretractable models for examining the role of PrPcat both the behavioral and cellular level.

P.58: Recombinant mouse prion proteinalone or combined with detoxified

lipopolysaccharide affects colon immuneprotein levels of FVB/N mice

Dagnachew Hailemariam, Tran Lam,Elda Dervishi, Grzegorz Zwierzchowski,

Suzanna Dunn, David Wishart, andBurim Ametaj


Cellular prion protein (PrPC) plays an impor-tant role in assembling tight junctions and bar-rier functions in the GI tract. The objective ofthis study was to evaluate whether recombinantmoPrP alone or combined with detoxified LPSwould affect concentrations of Slpi, Tlr4,CAMP, and Sprn in the colon under an in vitroUssing chamber system. Colons were selectedfrom 8 male FVB/N mice and were randomlyassigned to 6 treatments: (1) pyrogen free H2Oon the mucosal side (CON), detoxified lipopoly-saccharide on the mucosal side (D-LPS/M),mouse recombinant prion protein on the muco-sal side (moPrP/M), detoxified LPS on the sero-sal side (D-LPS/S), moPrP and D-LPS on


mucosal side (moPrP C D-LPS/M), moPrP onmucosal side and D-LPS on serosal side(moPrP/M C D-LPS/S). After euthanization,colon tissue was immediately excised andmounted between the mucosal and serosal reser-voirs of easymount Ussing diffusion chambers.After 40 min colon tissues were removed andstored at -80�C until analysis. Concentrations ofproteins in colon samples were determined usingcommercially available ELISA kits. Resultsindicated that concentrations of Slpi in moPrPCD-LPS and moPrP/M C D-LPS/S treatmentgroups were greater than CON group. CAMPprotein was decreased in the moPrP/SC D-LPS/S group as compared to CON samples, whereasconcentrations of Tlr4 were decreased by D-LPS and PrP/M and PrP/MCD-LPS/M; andSprn was lowered by D-LPS, PrP/M, and PrP/MCD-LPS but increased by PrP/MCD-LPS/S.Further research is needed to understand theimportance of these protein modifications in thecolon by moPrP and D-LPS.

P.59: Mouse recombinant prion proteinalone or in combination with bacterialLPS modulated production of colonproteins involved in innate immune

responses in FVB/N mice

Elda Dervishi, Grzegorz Zwierzchowski,Dagnachew Hailemariam, TH Lam,Suzanna Dunn, David Wishart, and

Burim Ametaj


The objective of the present study was totest whether mouse cellular prion protein(moPrPC) alone or in combination with lipo-polysaccharide (LPS) would alter innateimmunity proteins in the colon. Eight FVB/N male mice were used to collect colon sam-ples, which were mounted into a Ussingchamber. Chambers on both sides of theUssing chambers were filled with 7 mL ofKrebs buffer. The mucosal side of the cham-bers were supplemented with 700 mL ofpyrogen-free H2O (CON). Treatment 1

consisted in adding 700 mL of a solutioncontaining 100 mg/mL of lipopolysaccharide(LPS) from E. coli O111:B4 strain on themucosal side of the chamber. Treatment 2with 700 mL of a solution containing 100mg/mL of recombinant mouse (mo)PrPC (29–231) on mucosal side (PrPM). Treatment 3contained moPrPC C LPS at the 100 mg/mLon the mucosal side of the colon (PrPM CLPSM). The last treatment contained moPrPC

on the mucosal side and LPS on the serosalside of the chambers (PrPM C LPSS). Sam-ples were collected at 40 min of the initia-tion of the experiment and CAMP, SLPI,TLR4, and SPRN in the colon were mea-sured. CAMP was significantly greater inPrPM treatment compared to PrPM CLPSM. Concentration of SLPI wereincreased by PrPM, LPSM, PrPM, and LPSScompared to the CON group. Concentrationsof TLR4 were lowered by PrPM, LPSM, andtheir combination vs CON. LPS on the sero-sal side lowered levels of TLR4. SPRN con-centration was lower in PrPM and PrPM CLPSM.

P.60: New [PSIC]-no-more mutation inSUP35 with strong inhibitory effect on

[PSIC] propagation

Stanislav Bondarev, Lavrentiy Danilov,and Galina Zhouravleva

Saint Petersburg State University; Saint Petersburg,

Russia

Prions can be defined as infectious or inher-ited protein agents which propagation is basedon the ability to induce conformational conver-sion of cellular polypeptides. [PSIC] factor isone of well-studied yeast prions. Previously weconstructed 5 SUP35 alleles coding the proteinswith QQ or QN to KK substitutions within oneof the oligopeptide repeats (Bondarev et al.,2013). Positions and type of mutation werechosen according to the model of superpleatedb-structure proposed by Andrey Kajava. Twoof such alleles led to [PSIC] prion loss but bydifferent molecular mechanisms. Recently we


obtained a new mutation within another oligo-peptide repeat tht we not examined and probedits effect on prion stability. This allele, namedsup35-M0 according number of repeat, elimi-nated [PSIC] very efficiently even in presenceof SUP35. The prion loss reached up to 97%that exceeded the effect of PNM2 (less 20%)and sup35-M2 described previously (approxi-mately 40%). Nevertheless overexpression ofsup35-M0 promoted [PSIC] induction and thecorresponding protein formed fibrils in vitro.Our data provide experimental proof ofsuperpleated b-structure model with assump-tion about polymorphism of prion structuresand underlying role of N-terminal region ofSup35p in [PSIC] prion propagation.

The authors acknowledge Saint PetersburgState University for research grants1.37.291.2015, 0.37.696.2013, Russian Foun-dation of Basic Research for a research grant14-04-32213 and RRC MCT SPbSU.

P.61: Investigating amyloid-b oligomerand fibril dynamics using a novel

amyloid-b oligomer-specific antibody

Judith M Silverman, Ebrima Gibbs,Catherine M Cowan, and Neil R Cashman

University of British Columbia, Vancouver, Canada

Oligomers of the amyloid-b (Ab) peptideplay key roles in neurotoxicity and region-to-region spreading of Alzheimer’s disease (AD)neuropathology. We have produced a monoclo-nal antibody against a novel Ab epitope that isexposed only when Ab is in oligomeric form.The epitope consists of a constrained turn atresidues 26–28: cyclized serine-asparagine-lysine (cSNK). We have shown, by dot blot-ting, atomic force microscopy, and electronmicroscopy, that our antibody (5E3) binds tosynthetic Ab oligomers, but does not recognizemonomeric or fibrillar Ab. Furthermore, 5E3specifically recognizes Ab oligomers in brainsof AD patients and AD model mice. Our recentwork demonstrates that 5E3 is a promisingagent for immunotherapy to reduce toxic Aboligomers in the brain. However, we show here

that 5E3 also provides a useful tool to investi-gate in vitro by electron microscopy the mecha-nisms and time course of Ab oligomer andfibril formation. Additionally, we show that5E3 can reduce in vivo propagation of Abpathology to the mouse cortex following seed-ing by injection into the hippocampus. Thisdata is consistent with the hypothesis that Aboligomers displaying the cSNK epitope consti-tute the seed species of AD pathology. 5E3 canalso be used as a tool to investigate the mecha-nism of seed propagation: we find that the Aboligomers displayed on the surface of extracel-lular vesicles from AD mouse brains, whichmay participate in propagation, expose thecSNK epitope. These investigations show that5E3 is a promising reagent for both therapydevelopment and basic mechanism inquiry.

P.62: Combining anti-prion compoundswith complementary mode of action for

achieving additive effects in vitroand in vivo

Tazrina Alrazi and Hermann Schatzl

Department of Comparative Biology &

Experimental Medicine; Faculty of Veterinary

Medicine; University of Calgary; Calgary, Canada

The quest for therapeutic strategies in priondiseases did not yet result in drug regimenswhich are effective when used in prion-infectedhuman beings. This may surprise as a hugevariety of chemical compounds have beenreported as showing rather promising anti-prionactivities in vitro and to, although much less,extent also in animal models. Possible explana-tions might be that most compounds fail tocross the blood-brain-barrier at therapeutic con-centrations and that increasing the blood con-centration usually results in severe side effects.Successful animal models often use experimen-tal paradigms which are based on post-exposureand extra-CNS effects, and therefore do notreflect the real situation in human therapy. Ourrational is to use a combination of chemicalcompounds which showed certain anti-prioneffects already in vitro and in vivo when usedalone and from which the underlying molecular


mechanisms are well characterized. Given this,we can combine drugs with complementarymolecular targets and potentially additiveeffects. Presently, we test drugs which targetprion biogenesis (e.g. targeting PrPc substrate)or prion clearance (e.g., autophagy inducingdrugs) in prion-infected cultured cells at non-toxic concentrations. In parallel, selected com-binations are used in mouse models. Usingsuch a strategy we want to achieve additive oreven synergistic anti-prion effects, allowing usto lower the needed concentrations and associ-ated side effects in vivo. More refined futurecombination therapies might help to bridge theobvious gap between in vitro and in vivo resultsand provide a foundation for effective drugtherapies against prion diseases.

P.63: Analysis of activation state ofastrocytes with the progression of

prion diseases

Minori Kuroda, Takeshi Yamasaki,Akio Suzuki, Rie Hasebe, and

Motohiro Horiuchi

Hokkaido University, Sapporo, Japan

Activation of astrocytes and microglia is oneof the hallmarks in prion diseases. Data on theinvolvement of microglia in pathogenesis ofprion diseases are accumulating; however therole of astrocytes in prion diseases is not wellunderstood. To elucidate the involvement ofastrocytes in pathogenesis of prion diseases, weanalyzed activation states of astrocytes inprion-infected mouse brains. We establishedthe method for isolation of astrocytes frommouse brains using magnetic activated cellsorting (MACS) with Anti-Astrocyte Cell Sur-face Antigen-2 (ACSA-2) antibody. Purity ofastrocytes was analyzed by flow cytometricanalysis and TaqMan assay. Eleven genes ofcytokines, chemokines, neurotrophic factorsand their receptors that are reported to be up-regulated in prion diseases and other neurode-generative diseases were analyzed by TaqMangene expression assay. Flow cytometric analy-sis showed that the ACSA-2-bound fraction

contained approximately 95% of ACSA-2-posi-tive cells and that contamination of CD11b-positive microglia and CD31-positive endothe-lial cells was less than 0.5%. ACSA-2-positivecells included GFAP (astrocytes marker)-posi-tive and negative cells, whereas ACSA-2-nega-tive cells included only GFAP-negative cells.This suggests the utility of ACSA-2 for astro-cytes isolation. High gene expression of GFAPbut a trace of CD11b and CD31 gene expres-sions also demonstrated the successive isola-tion of astrocytes by anti-ACSA-2 antibody.Among genes tested, CXCL10 chemokine geneexpression was upregulated from 90 dpi to 120dpi, whereas expression of remaining 10 genesshowed no remarkable differences. The tempo-ral up-regulation of CXCL10 gene suggests thatactivation state of astrocytes changes with thedisease progression.

P.64: Behavior of antiprion smallmolecules in RT-QuIC

Sonia Vallabh1,2, Eric Minikel1,2, andByron Caughey3

1Prion Alliance, Cambridge, MA, USA; 2Harvard

Medical School, Boston, MA, USA; 3Laboratory of

Persistant Viral Diseases; NIAID; Rocky Mountain

Laboratories, Hamilton, MT, USA

The in vitro prion seeding reaction known asreal-time quaking induced conversion (RT-QuIC) has proven its utility as a highly sensi-tive and specific diagnostic tool [1]. Initialefforts have been made to explore RT-QuIC’spotential to inform drug discovery [2,3], butthe relationship between observed antiprionactivity in RT-QuIC and potential for efficacyin vivo remains inconclusive. We systemati-cally query this relationship by leveraging com-pounds with established antiprion activity invivo. To date, though no small molecules haveyet been reported as effective against a humanprion strain [4,5], 3 orally bioavailable smallmolecules have been reported to dramaticallyextend survival time in mice infected withRML prions: IND24, cpd-b and anle138b[4,6,7]. These and many other compounds are


active in phenotypic assays, reducing the for-mation of proteinase K-resistant PrPSc in RML-infected cultured cells, but the lack of humanprion-infected cell lines precludes applicationof these assays to human prion strains. Here weassess the ability of RT-QuIC to detect theestablished antiprion activity of these com-pounds in vitro. This approach could help todecipher the still elusive mechanism of actionof these compounds, and if successful could beextended to testing candidate compoundsagainst human prion strains.

P.65: Myotube cultures as a prion sourcefor structural investigations

M Carmen Garza1,2, Pamela Banser1,3,Leonardo Cortez1,4,

Jacques van der Merwe1,4,David A Kramer2, Nathalie Daude1,4,Richard Fahlman2, Valerie Sim1,4,David Westaway1,4, Judd Aiken1,5,

Debbie McKenzie1,3, and Holger Wille1,2


University of Alberta; Edmonton, Canada;2Department of Biochemistry; University of

Alberta; Edmonton, Canada; 3Department of

Biological Sciences; University of Alberta;

Edmonton, Canada; 4Department of Medicine;

Edmonton, Canada; 5Department of Agriculture;

Food and Nutritional Science; Edmonton, Canada

Our knowledge about the prion diseases con-tains important gaps that need to be filled: E.g.the structure of the infectious isoform of theprion protein (PrPSc) and the conversion pro-cess from its cellular precursor (PrPC). Mosttechniques to decipher the structure of PrPScare hampered by its insolubility and the needfor substantial amounts of highly purified pro-tein. Nearly all approaches that address thestructure of PrPSc rely on brain samples frominfected animals. In our current study we aretrying to overcome the limitations of thissource by using prion-infected C2C12 myo-tubes. Myotubes are non-proliferative cellsthat, upon infection with RML prions, can gen-erate high levels of PrPSc and infectivity, about

10 times more than other mammalian cells inculture (e.g., N2a cells) (Herbst et al., 2013;PLoS Pathogens; 9:e1003755). We encoun-tered difficulties in the purification of myotube-derived PrPSc via precipitation with sodiumphosphotungstate due to the co-precipitation offibronectin, fibrillin, and collagen; these extra-cellular matrix proteins were identified by massspectrometry and validated through Westernblotting. In OptiPrep gradients (sedimentationvelocity and sedimentation equilibrium gra-dients), these proteins co-migrated not onlywith some fractions of myotube-derived PrPSc,but also with PrPC. Fortunately, fractionationof the OptiPrep gradients achieved a completeseparation of PrPSc from the extracellularmatrix proteins in many fractions, and providedadditional information about the density andhydrodynamic properties of the PrPSc aggre-gates. Electron microscopy will be used to eval-uate the quaternary structure of the PrPScaggregates and their overall suitability forstructural studies.

P.66: Transport of CWD prions inAlberta soils

Alsu Kuznetsova1, Debbie McKenzie2,Tariq Siddique1, and Judd Aiken2

1University of Alberta; Department of Renewable

Resources; Edmonton, Canada; 2University of

Alberta; Centre for Prions and Protein Folding

Diseases; Edmonton, Canada

The transmission of chronic wasting disease(CWD) includes environmental pathways, par-ticularly soils as disease reservoirs. Soils differdramatically in their capacity to adsorb PrPCWD

due to differences in mineral composition,humus content and particle surface area. Min-eral and organic compounds have the ability tobind PrPCWD impacting infectious properties.The extreme variability of these soil constitu-ents suggests that the PrPCWD fate and behaviorwill depend on specific soil properties. The soilmoisture regime also has the potential to affecttransportation of compounds through a soil pro-file. PrPCWD can be bound to soil particles with


3 hypothetical scenarios for prion fate: (i)prions stay in the surface soil horizon andremain bioavailable for grazing animals; (ii)prions can be transported into lower soil hori-zons and become unavailable for consumption;or (iii) prions can migrate through the soil pro-file and end up in ground water. We performedbench-scale experiments with soil columns toevaluate the potential for transportation ofPrPCWD using soils from different regions ofAlberta, Canada. The Luvisols found in north-ern Alberta have an ustic/udic moisture regimeand illite as a predominant clay mineral. Theprion binding capacity of illite is poor suggest-ing it cannot contribute to prion binding andPrPCWD can migrate through the soil profile.Chernozems are found in the CWD-endemicregion in southern Alberta and have an aridicsoil moisture regime, high amount of humuscontent and contain montmorillonite. In theChernozem soil columns PrPCWD remains onthe soil surface and does not migrate in lowerhorizons.

P.67: CWD prion infection ofdifferentiated neurosphere cultures

Yoshifumi Iwamaru1,2,Candace Mathiason1, Glenn Telling1, and

Edward Hoover1

1Prion Research Center; Department of

Microbiology; Immunology and Pathology; College

of Veterinary Medicine and Biomedical Sciences;

Colorado State University; Fort Collins, Colorado

USA; 2Influenza and Prion Disease Research

Center; National Institute of Animal Health;

Tsukuba, Japan

Most of the cell culture models of prioninfection are based on rodent cell lines foundto be permissive to rodent-adapted prions. Inorder to develop more diverse cell culturesystems permissive to infection with natu-rally occurring prions in humans and ani-mals, one possible strategy is use of neuralstem cell cultures from transgenic (Tg)mouse lines expressing prion protein (PrP)of the native host species. Neural stem cells

are self-renewing, multipotent progenitorsthat can be cultured as suspended cell aggre-gates termed neurospheres. Building on ourprevious work with mouse-adapted prions,we aimed to establish a cell culture modelthat would support replication of non-adapted cervid-origin CWD prions by usingdiffferentiated neurosphere cultures from cer-vid PrP expressing Tg mice.

Primary neurosphere cultures were iso-lated from brains of neonatal PrP-null miceand Tg mice expressing cervid PrP-E226(Tg5037 mice). The neurosphere cultures(NP0 and NP5037, respectively) wereexpanded by passage >10 times in serum-free media supplemented with N-2 factors,epidermal growth factor (EGF) and basicfibroblast growth factor (bFGF) and then dif-ferentiated by withdrawal of i) EGF ii)bFGF iii) both bFGF and EGF with addingfetal bovine serum under adherent cultureconditions. Upon infection with brain homo-genates from CWD positive white-tailed deeror elk, only the EGF-withdrawn cultures(dNP5037/bFGF) accumulated substantiallevels of proteinase K-resistant PrP (PrPres).Importatntly, PrPres was not detectable ininfected dNP0/bFGF cultures, indicatingdNP5037/bFGF cultures supported de novoPrPres formation. The neurosphere cell cul-ture model offers an alternative approach forprion infectivity assays in various species.

P.68: Inter-domain structure in thecopper-bound cellular prion proteinrevealed by site-directed spin labeling

and EPR spectroscopy

Eric GB Evans1, M Jake Pushie2, andGlenn L Millhauser1

1University of California; Santa Cruz, CA USA;2University of Saskatchewan; Saskatoon, SK,

Canada

The cellular prion protein (PrPC) is amembrane-anchored glycoprotein consistingof 2 domains: a flexible N-terminal domainthat participates in metal binding, and a


mainly helical C-terminal domain that con-verts to b-sheet structure in the course ofprion disease. These two domains have tradi-tionally been thought of as non-interacting;however, recent cellular and biophysical evi-dence has forced a reconsideration of thisview. We recently reported a novel tertiaryfold in which the Zn2C-bound octarepeatdomain contacts the exposed surface of heli-ces 2 and 3.1 The apparent stability of thisinteraction was diminished in several mutantPrPs that result in familial prion disease,suggesting a potential role for inter-domainstructure in disease progression. In the pres-ent work, we examine inter-domain structurein Cu2C-bound recombinant PrP using site-directed spin labeling of the geneticallyencoded unnatural amino acid pAcPhe andelectron paramagnetic resonance (EPR) spec-troscopy. Distance measurements betweenCu2C, bound with high affinity to the octare-peat domain, and spin labeled residues of theglobular C-terminus reveal that the copper-bound octarepeats interact with a negativelycharged surface defined by helices 2 and 3.Our results are supported by moleculardynamics simulations and indicate that thiscis interaction is stabilized by electrostatics.Our findings suggest that metal-induced ter-tiary structure may be a general property ofPrPC, and that disruption of this interactionmay be a contributing factor in prion diseasepathology.

References

1. Spevacek AR. Evans EG, Miller JL, Meyer HC, Pelton

JG, Millhauser GL. Structure 2013; 21:236;

PMID:23290724; http://dx.doi.org/10.1016/j.str.2012.

12.002

P.69: Distinct pathological phenotypes ofCreutzfeldt-Jakob disease in recipientsof prion-contaminated growth hormone

Ignazio Cali1,2, Cathleen Miller3,Tetsuyuky Kitamoto4, Joseph Parisi5,

Michael Geschwind6, Pierluigi Gambetti1,and Lawrence Schonberger7

1National Prion Disease Pathology Surveillance

Center (NPDPSC); Department of Pathology; Case

Western Reserve University; School of Medicine;

Cleveland, OH USA; 2Department of Clinical and

Experimental Medicine; Second University of

Naples; Naples, Italy; 3Kaiser Permanente

Vancouver Medical Center; Vancouver, WA USA;4Graduate School of Medicine; Tohoku University;

Sendai, Japan; 5Departments of Laboratory

Medicine & Pathology and Neurology; Mayo

Clinic; Rochester, MN USA; 6Department of

Neurology; Memory and Aging Center; University

of California; San Francisco, CA USA; 7National

Center for Emerging and Zoonotic Infectious

Diseases; Centers for Disease Control and

Prevention; Atlanta, GA USA

The peripheral administration of growth hor-mone (GH) from prion-contaminated cadavericpituitary glands is believed to be causative ofiatrogenic Creutzfeldt-Jakob disease (iCJD) inmore than 225 subjects worldwide. The presentstudy describes the neuropathology and molec-ular features of 3 of the 30 identifiediCJD cases among the approximately7,700 recipients of cadaveric pituitary hormonein the US National Hormone and Pituitary Pro-gram (NHPP). All three cases were methionine(M) homozygous at codon 129 of the prion pro-tein (PrP) gene (GH-CJDMM) and all receivedNHPP hormone produced before 1977 when anew hormone purification protocol was intro-duced that reduced the risk of prion contamina-tion. Neuropathological examination revealeddivergent phenotypes. The first phenotype,observed in the most recent US NHPP GH-CJDcase, was characterized by the presence of amy-loid plaques and reminiscent of sCJDMV2-Kand, to some extent, variant CJD (vCJD). Thesecond phenotype showed no plaques andshared several, but not all, characteristics with


the sCJDMM(MV)1 subtype. However, PK-resistant PrPSc (resPrPSc) from GH-CJDMMco-migrated with resPrPSc type 1 (GH-CJDMM1) of sCJDMM1, but not with type 2of sCJDMV2-K. Histopathological phenotypeswith or without plaques also have beendescribed in 2 groups of Japanese dura mater(d) graft-associated CJD (dCJD) with the same129MM genotype but apparently different gelmobility of resPrPSc type 1. Our study suggeststhat phenotypic diversity in these iatrogenicdiseases reflects adaptation of different exoge-nous prion strains to the 129MM host and/or todifferent locations of the initial PrPC to PrPSc

conversion.

P.70: Experimental transmission ofchronic wasting disease to sheep

and goats

Gordon Mitchell, Nishandan Yogasingam,Ines Walther, and Aru Balachandran

National and OIE Reference Laboratory forScrapie and CWD; Canadian Food Inspection

Agency; Ottawa, ON, Canada

The persistence of chronic wasting disease(CWD) in North American cervids, coupledwith efforts to eradicate scrapie in sheep andgoats, necessitates an understanding of thetransmission, clinical and diagnostic character-istics of CWD in small ruminants. Oral andintracerebral transmission studies were con-ducted in sheep and goats using tissues fromCWD-infected elk. Four lambs and 4 goatswere orally inoculated with a pooled brain andlymph node homogenate from a group offarmed elk with clinical CWD. At study end-point, there was no evidence of primary CWDtransmission in the sheep or goat tissues exam-ined by ELISA, western blot and immunohis-tochemistry (IHC). Two lambs which werechallenged intracerebrally with the samepooled elk inoculate displayed neurologicalsigns beginning at 27 months postinoculation(mpi) and were euthanized within 10 d of eachother at 28 mpi. Testing of tissues by ELISAand IHC confirmed disease transmission and

revealed differences in the distribution andintensity of PrPd deposition between animals.Western immunoblot analysis identified char-acteristics permitting the differentiation ofCWD in sheep from other prion diseases insmall ruminants. CWD-infected tissue from theintracerabrally-inoculated sheep has undergonesecondary passage into sheep and goats andcurrently shows no evidence of oral transmis-sion in rectal mucosa biopsies at 20 mpi. Thesefindings corroborate evidence of a significantspecies barrier preventing the oral transmissionof CWD to sheep and goats, and identify diag-nostic characteristics to enable the differentia-tion of prion diseases affecting smallruminants.

P.71: Predicting new prion candidatesin yeast

Jenifer Shattuck, Aubrey Waechter, andEric Ross

Colorado State University, Fort Collins, CO USA

Prions are infectious proteins capable of self-propagating and transmitting between organ-isms. Even though there is no homolog to themammalian prion protein in yeast, several solu-ble proteins can form heritable aggregates denovo. These proteins provide a model systemto investigate the nucleation, aggregation andpropagation steps involved in the formation ofa prion fibril.

Several prion prediction algorithms havebeen developed to predict yeast proteins thathave the propensity to form prions. One ofthese algorithms was previously developed inour laboratory (Prion Aggregation PredictionAlgorithm, PAPA, Toombs et al., 2012).Therefore, we used PAPA to scan the yeast pro-teome to identify proteins that contain domainspredicted to have prion activity (prion-likedomains). These prion-like domains weretested in 4 prion activity assays to assess theiractivity in vivo as well as in vitro. Here we pro-vide the preliminary results from our in vivoprion activity assays. Using these preliminaryresults, we are currently investigating a couple


respective full-length proteins for prion activityby developing phenotypic assays.

Ultimately, we may identify new prion can-didates in yeast, which will contribute informa-tion about the parameters necessary for prionformation and insight into the functions prionsplay in yeast. In addition, by confirmingPAPA’s ability to predict prion proteins fromthe yeast proteome, it allows the possibility toapply this methodology to other proteomes.

P.72: Polymorphisms in prion proteinamino acid 170 do not alter the highsusceptibility of red-backed voles

(Myodes gapperi) to chronicwasting disease

Christina Carlson, Jay Schneider,Dennis Heisey, and Christopher Johnson

USGS National Wildlife Health Center, Madison,

Wisconsin, USA

The b2-a2 loop structure of the cellular prionprotein (PrPC) has been identified as a potentialdeterminant of a host’s susceptibility to chronicwasting disease (CWD). The NMR structures ofPrPC from species that possess an asparagine atposition 170 (170N; deer, elk, bank voles) showa b2-a2 loop that is more rigid than PrPC pos-sessing a serine at that same residue (170S;mice, cattle, humans). The 170N genotype isalso associated with CWD susceptibilitywhereas 170S appears to contribute to CWDresistance. We identified and captured a popula-tion of red-backed voles that display a naturalasparagine/serine polymorphism at position170. We bred these voles to produce pups withhomozygous (170SS, 170NN) or heterozygous(170SN) genotypes and intracerebrally-chal-lenged them with white-tailed deer (Odocoileusvirginanus) CWD. Following challenge, all 3genotypes of voles displayed high attack ratesof disease (100% for 170NN and 170SNcohorts, and 89% for 170SS cohort) and statisti-cally-indistinguishable survival times (333§ 28d post-infection (dpi), 334§ 53 dpi, and 349§30 dpi, respectively; median survival time §95% confidence interval). Clinical signs of

disease were similar across experimental chal-lenge groups and included lethargy, ataxia, anddecreased burrowing activity. The glycoformprofiles and deposition of the abnormal prionprotein were assessed by immunoblot andimmunohistochemistry analyses. Our work sug-gests that susceptibility to white-tailed deerCWD in red-backed voles is not reduced by thepresence of 170S in the b2-a2 loop of PrPC.

P.73: Oral challenge of goats withatypical scrapie

Silvia Colussi1, Maria Mazza1,Francesca Martucci1, Simone Peletto1,Cristiano Corona1, Marina Gallo1,Cristina Bona1, Romolo Nonno2,

Michele Di Bari2, Claudia D’Agostino2,Nicola Martinelli3, Guerino Lombardi3,

and Pier Luigi Acutis1

1Istituto Zooprofilattico Sperimentale del Piemonte;

Liguria e Valle d’Aosta; Turin, Italy; 2Istituto

Superiore di Sanit�a; Rome, Italy; 3IstitutoZooprofilattico Sperimentale della Lombardia e

dell’Emilia Romagna; Brescia, Italy

Atypical scrapie transmission has been dem-onstrated in sheep by intracerebral and oralroute (Simmons et al., Andreoletti et al., 2011)but data about goats are not available yet. In2006 we orally challenged four goats, fivemonths old, with genotype R/H and R/R atcodon 154. Animals died starting from 24 to77 months p.i. without clinical signs. They allresulted negative for scrapie in CNS andperipheral tissues using Western blot andimmunohistochemistry. Nevertheless thesegoats could still represent carriers. This hypoth-esis was investigated through bioassay in tg338mice, a sensitive animal model for atypicalscrapie infectivity. By end-point dilution titra-tion, the starting inoculum contained 106.8

ID50/g. In contrast, all tissues from challengedgoats were negative by bioassay.

These negative results could be explainedwith the low infectivity of the starting inocu-lum, which could have been unable to induce


disease or infectivity within our period ofobservation. However the challenge conditionscould have been a bias too: as the matter of thefact, while the oral challenge of classical scra-pie is still effective in sheep 6–10 months old(Andreoletti et al., 2011), Simmons et al.(2011) demonstrated a very short efficacyperiod for atypical scrapie (24 hours afterbirth), hypothesizing that natural transmissioncould occur mainly via milk. Our work sug-gests that this could be true also for goats and itshould be taken into account in oral challenges.However a low susceptibility of goats to atypi-cal scrapie transmission via oral route cannotbe excluded.

P.74: Transmission of experimentalCH1641 scrapie to wild-type mice

Lucien van Keulen1,*, Jan Langeveld1,Corry Dolstra1, Jorg Jacobs1, Alex Bossers1,

and Fred van Zijderveld2

1Department of Infection Biology; Central

Veterinary Institute of Wageningen UR, Lelystad,

The Netherlands; 2Department of Bacteriology and

TSEs; Central Veterinary Institute of Wageningen

UR, Lelystad, The Netherlands

Introduction. CH1641 was isolated in theUK in 1970 from a natural case of scrapie in aCheviot sheep and was further passaged intra-cerebrally in sheep. CH1641 has been the sub-ject of extensive research because of thebiochemical similarities of PrPres fromCH1641- and BSE-affected sheep brains. Pre-vious attempts to transmit CH1641 to wild typemice have been unsuccessful. We report herefor the first time, the positive transmission ofexperimental CH1641 to RIII mice and com-pare the incubation period, PrPSc profile andPrPres Western blot properties to those ofknown scrapie and BSE reference strains.

Methods. The CH1641 brain homogenateused in this study came from a pool a 5 sheepbrains which had been challenged intracere-brally with brain material from the third

passage of CH1641 in sheep. Groups of 15–20RIII mice were inoculated intracerebrally witha 10% brain homogenate of CH1641. Thebrains of the mice were examined by PrPSc pro-filing and triplex Western blot as reportedpreviously.

Results. Surprisingly CH1641 transmitted toRIII mice with a 100% attack rate althoughwith a long incubation period (794 § 149 d).The resulting PrPSc profile was unlike any ofthe profiles of the scrapie and BSE referencestrains reported previously. Triplex Westernblot pointed after first passage to a very lowPrPres level. We observed a reduction of molec-ular mass of the non-glycosyl PrPres moiety andconcomittant N-terminal 12B2 epitope signal.In comparison to the original CH1641 inocu-lum there was a lack of a dual population ofPrPres.

P.75: Development of pre-mortemdiagnosis for suspected Creutzfeldt-

Jakob diseases’ patients

SuYeon Kim, JaeWook Hyeon,YeongRan Ju, JiYeon Lee, WonCheol Lee,

and YeongSeon Lee

Korea NIH (KCDC); Cheonju, Chungcheongbukdo,

Republic of Korea

Creutzfeldt-Jakob disease (CJD) is the mostrepresentative human prion disease caused byabnormal accumulation of misfolding prionprotein. The diagnosis is performed with fea-tures of magnetic resonance imaging, electro-encephalogram and elevated the 14-3-3 proteinfindings, prion protein gene polymorphisms. Inlaboratory, the protein detection and analysis ofthe gene polymorphisms have been monitored,and then clinicians determined as CJD patientor not CJD case combining specific clinicalopinions in Korea. We aimed evaluate the epi-demiological tendency, and the possibility ofearly diagnosis through the application of clini-cal features included the protein tests andgenetic analysis. We detected 14-3-3 protein,and analyzed PRNP genotypes for suspected


cases (2010–2014). The results were combinedwith progressive dementia, myoclonus, andmemory decline, and their relationships wereanalyzed. They were almost within the agerange of 60–80 years, and the numbers of maleand female were similar. Approximately 49%showed positive for 14-3-3 protein, and thepolymorphisms reported to genetic pathogenicfactor inherited CJD showed in 11 patients.Three definite and 14 possible sCJD patientsdefined except for one were positive for 14-3-3,and several probable sporadic cases had patho-genic genetic factors like P102L, E200K andV180I. The clinical presentations showed pro-gressive dementia, visual illusion, myoclonus,ataxia, akinetic mutism, and memory decline.Some MRI and EEG findings showed high sig-nal abnormalities in the fronto-temporal cortexand typical periodic sharp wave complexes.We consider that the active following surveil-lance for patients would be added to improvethe specificity of early CJD diagnosis.

P.76: Deciphering the molecularchaperone network using yeast prions

Michael Reidy and Daniel Masison

Laboratory of Biochemistry and Genetic; National

Institute of Diabetes Digestive and Kidney

Diseases; National Institutes of Health; Bethesda,

MD USA

Since the discovery in 1994 that known butunexplained non-Mendelian phenotypes in Sac-charomyces cerevisiae were due to prions,much work has shown the reliance of prionpropagation on molecular chaperones. Yeastprions propagate as infectious amyloid fiberscomposed of misfolded forms of cellular pro-teins. Replication of these fibers in yeast cellsrequires new seed generation via breakagemediated by the cellular protein disaggregationsystem. In recent years we have learned muchabout the components and mechanisms of vari-ous prion-related processes. The various prionsrely on the chaperone machinery to differentdegrees. For example, elevation of the Hsp40Ydj1 causes curing of the [URE3] prion but has

no effect on [PSIC]. We recently showed ele-vated Ydj1 reduced availability of a differentHsp40 isoform, Sis1, which is relied upon moreby [URE3] than [PSIC]. Thus, subtle changesin the chaperone landscape lead to measurablechanges in the maintenance of prions that maynot be observed using alternative assays. In thisway using the yeast prion system has producedsignificant insight into the important functionaldistinctions that exist between nearly identicalchaperone isoforms. These observations haveled to new appreciation for the notion thatchaperone isoforms that were once thought tobe merely redundant in function also performunique tasks. Here, we highlight several of ourrecent findings demonstrating the power andutility of the yeast prion system for studyingfunctions, specificity and cooperation of chap-erone machinery components.

P.77: Pre-clinical biomarkers ofprion infection

Danielle Gushue1, Allen Herbst1,Lingjun Li2, Debbie McKenzie1, and

Judd Aiken1


University of Alberta; Edmonton, Canada;2Department of Chemistry; University of Wisconsin,

Madison; Madison, WI USA

KEYWORDS. Creutzfeldt-Jakob disease, ante-mortem, biomarkers, 14-3-3, neuron-specificenolase

An estimated 1/ 2000 people in the UK areasymptomatic carriers for variant Creutzfeldt-Jakob disease (vCJD), demonstrating a need forscreening and early detection. Ante-mortemtesting of CJD is currently performed uponclinical presentation of the disease. Surrogatemarkers are used in combination with othermethods for differential diagnosis of CJD; how-ever, existing markers have limitations. Theobjective of these studies is to define the pre-clinical abundance of known prion disease bio-markers as well as identify novel pre-clinical,ante-mortem markers of prion disease. We


adapted prion disease to rats, facilitating aproteomic approach to a bioavailable fluid(CSF) at preclinical and clinical disease stages.This contrasts with human samples, which aregenerally only available at clinical stage. Therat CSF proteome was compared betweeninfected rats and age-matched controls throughmass spectrometry. A number of proteins up-regulated and/or specific to prion disease wereidentified. These proteins included CJD bio-markers, 14-3-3s and neuron-specific enolase(NSE), demonstrating the utility of using ratprion disease for pre-clinical biomarker identi-fication. The abundance of these known bio-markers during preclinical stages of the diseasewill be presented. Since the composition ofCSF reflects the pathological processes of thebrain, tracking the progression of prion infec-tion in rats will allow us to further define priondisease neurodegeneration.

P.78: Characterization of interactionsbetween the cellular prion protein(PrPC) and toxic assemblies of the

amyloid-b peptide

Erin Bove-Fenderson and David Harris

Boston University School of Medicine; Boston,

MA USA

Alzheimer disease (AD) is a devastatingneurodegenerative disease that affects millionsof people worldwide, and presents one of ourbiggest challenges in addressing the healthneeds of an aging population. Today, multiplelines of research aim to achieve methods forearly diagnosis and treatment, as well as a basicunderstanding of the biology involved in thedisease process. Two pathological hallmarks ofAD are plaques, comprised primarily of amy-loid-b (Ab) peptide, and neurofibrillary tangles,comprised of the micro-tubule associated pro-tein Tau. Our understanding of how these hall-marks are related to neurodegeneration is stillincomplete. However, soluble oligomers of Ab,which collect prior to or during plaque forma-tion, have been shown to negatively influencecell signaling and synaptic function in vitro.

Several receptor targets have been found tolink oligomers with cell toxicity, one of whichis the cellular prion protein (PrPC). We hypoth-esize that PrPC can be used to capture and char-acterize specific toxic oligomers of Ab, leadingto a better understanding of these oligomersand how they interact with PrPC to promoteneurodegeneration. Previous studies haveimplicated mid-size aggregates (10–20mers) orrelatively large protofibrils of Ab as bindingpartners for PrPC. Our study sheds light on thelimitations involved in working with in vitropreparations of soluble Ab oligomers, andexamines more rigorously the characteristics ofthe PrPC-Ab complex through the use of multi-ple surface and solution techniques, includingSPR and fluorescence anisotropy.

P.79: Alpha-Synuclein: A potentialCerebrospinal fluid biomarker fordifferentiation of CJD from AD

Mohsin Shafiq, Franc Llorens,Saima Zafar, and Inga Zerr

Clinical Dementia Center; Department of

Neurology and Psychiatry; University Medical

Center Goettingen (UMG); G€ottingen, Germany

Alpha-Synuclein (Syn-a) is one of theabundant proteins in synapses and itsanomalies are associated with varioussynucleinopathies. Synaptic damage may leadto release of Syn-a in CSF;1 this release maybe prominent in rapid neurodegeneration e.g.CJD and Alzheimer disease through rapidprogression (rpAD). Current study involvesmeasurement of CSF Syn-a in sporadic CJD(sCJD) and AD with aim to test CSF Syn-apotential for differential clinical diagnosis.CSF Syn-a in sCJD and AD patients againstnon-dementia controls were measured usingconventional ELISA and an electro-chemiluminescence based system developedusing Meso-Scale DiscoveryTM (MSD) ELISA-plates. Conventional ELISA revealed 2 folds(P � 0.05) elevated CSF Syn-a in CJD-MM1(age D 65.3 § 9.1 year), in comparison to agematched controls (age D 69.20 § 9.5 year).


Non-significant differences were noticed inVV2 (age D 64.2§ 10.4 year) in comparison tocontrols. CSF Syn-a from rpAD cases (age D65.8§ 8.6y) were also compared to AD (age D69.8 § 8.9 year) and controls (age D 66.6 §12.9 year), but no significant differences werenoticed. MSD-assay exhibited highersensitivity than conventional ELISA; indiscriminating control and sCJD groups (ROC-AUC of 0.9408 and 0.8435 respectively). CSFSyn-a in sCJD group was noticed to be 6.7folds higher than control samples (P � 0.0001).In addition, Syn-a levels were unchanged inAD compared to control cases. In conclusion,our data showed altered level of Syn-a in CSF;and Syn-a can be used as differentiating indexfor sCJD and AD cases, in contrast to classicalAD cases from those with rapid progressioncourse.

KEYWORDS. Alpha-Synuclein, AD, sCJD,Cerebro-spinal fluid, ELISA, Neuro-degeneration

Reference

1 El-Agnaf, et al. FASEB J 2003; PMID:14519670

P.80: Predicting prion propensity inhuman proteins

Sean Cascarina and Eric Ross


In humans only a single prion-forming pro-tein named PrPc (for “cellular prion protein”)is currently known, yet many more neurode-generative disorders involve aberrant proteinaggregation. The classical model for these dis-eases has involved cell-autonomous aggrega-tion, assuming that aggregation occursindependently in each cell within a diseasedpatient. However, more recent models haveproposed a non-cell-autonomous progression ofdisease in which aggregates formed in one cellmay be transmitted to neighboring cells. Theseaggregate seeds then cause aggregation of thesoluble protein in the “infected” cells, similar

to the prion diseases. Within the past few years,a number of proteins that exhibit prion-likeaggregation and spread to neighboring tissueshave been discovered in patients with Amyo-trophic Lateral Sclerosis (ALS). Although ALShas been studied for a number of decades, theseproteins were only recently linked to ALS bychance. This demonstrates a clear need for anaccurate method to systematically identifyadditional proteins that may play a pathologicalrole in neurodegenerative disorders. Takingadvantage of the compositional similarity ofthese proteins to the known yeast prions, I planto use the prion prediction methodology thatour lab has pioneered to develop an entirelynew algorithm specifically suited for this classof neuronal proteins.

P.81: Optimization of liposomes for invivo delivery of PrPC siRNA to the brain

Heather Bender and Mark Zabel


Prion diseases, or Transmissible SpongiformEncephalopathies (TSEs), primarily affectsheep, cattle, cervids, and humans. The emer-gence of prion diseases in wildlife populationsand the increasing impact of prion diseases onhuman health has led to an increase in the studyof antiprion compounds. Recent studies havefound antiprion compounds that can inhibit theinfectious prion isomer (PrPRes) or down regu-late the normal cellular prion protein (PrPC).These compounds are often found through thescreening of drug or chemical compound librar-ies. However, most of these chemicals cannotcross the blood brain barrier to effectivelyinhibit PrPRes formation in brain tissue or tospecifically target neuronal PrPC. Also, thesecompounds tend to have multiple off targeteffects, and are often too toxic to use in animalor human subjects. Therefore, we have pro-posed using intravascular siRNA that is tar-geted toward PrPC as a safer and more effectiveantiprion compound. To protect the siRNAfrom serum degradation and RES elimination,we have encapsulated it within anionic


PEGylated liposomes using protamine sulfate.Encapsulation of the siRNA with protaminesulfate results in »90% encapsulation effi-ciency as compared to 40–80% efficiency with-out protamine. We are also using the smallpeptide RVG-9r to target the siRNA to nico-tinic acetylcholine receptors within the CNS. Inorder to reduce the elimination of the peptide,we have covalently bonded it to the PEGgroups of the liposomes using carbodiimidereactions. In future experiments, we will deter-mine the effectiveness of this drug delivery sys-tem using flow cytometry.

P.82: Investigation of RNA-bindingproteins with prion-like domains

Amy Boncella, Brittani Watkins, andEric Ross


Recently, mutations in a number of RNA-binding proteins have been linked to variousneurodegenerative diseases. Many of these pro-teins are involved in stress granule and process-ing body (P-body) formation. Stress granuleand P-body levels increase when cells are sub-jected to stress and decrease once the stress iseliminated. Reversible aggregation of thesecomplexes is required for normal cellular func-tion; however, mutations can make some ofthese proteins more aggregation prone, causingaccumulation of protein aggregates. Several ofthese proteins appear to form these assembliesvia regions termed prion-like domains (PrLDs).These domains contain amino acid composi-tions similar to those of known prions. Our labis interested in developing the ability to predictprion and aggregation propensities of differentproteins in yeast. PAPA (Prion AggregationPrediction Algorithm) was designed by our labto predict the prion propensity of Q/N-richamino acid sequences. Using predictions fromthis algorithm, we are investigating how muta-tions made in the PrLDs of different RNA-binding proteins alter stress granule and P-bodydynamics in yeast. We are utilizing fluores-cence microscopy to monitor the formation of

aggregates in vivo and thus observe the effectsof different mutations. An understanding ofhow mutations affect the formation of stressgranules and P-bodies in yeast may help to elu-cidate the mechanisms of similar disease-rele-vant proteins in the future.

P.83: Gerstmann-Str€aussler-Scheinkerdisease with F198S mutation: Selectivepropagation of PrPSc and pTau upon

inoculation in bank vole

Michele Angelo Di Bari1, Romolo Nonno1,Laura Pirisinu1, Claudia D’Agostino1,Geraldina Riccardi1, Guido Di Donato1,Paolo Frassanito1, Bernardino Ghetti2,

Pierluigi Gambetti3, and Umberto Agrimi1

1Department of Veterinary Public Health and Food

Safety; Istituto Superiore di Sanit�a; Rome, Italy;2Indiana University-Purdue University

Indianapolis; Department of Pathology and

Laboratory Medicine; Indianapolis, IN USA; 3Case

Western Reserve University; Cleveland, OH USA

Gerstmann-Str€aussler-Scheinker disease withF198S mutation (GSS-F198S) is characterizedby the presence of PrP amyloid plaques as wellas neurofibrillary tangles with abnormally-phos-phorylated tau protein (pTau) in the brain. Therelationship between tau protein and PrP in thepathogenesis of GSS-F198S is unknown. In aprevious study, we inoculated intracerebrally 2GSS-F198S cases in 2 lines of voles carryingeither methionine (Bv109M) or isoleucine(Bv109I) at codon 109 of PrP. GSS-F198S trans-mitted rather efficiently to Bv109I, but not toBv109M.

Here we investigated the presence of pTau,as assessed by immunohistochemistry withanti-pTau antibodies AT180 and PHF-1, in thesame voles previously inoculated with GSS-F198S. Among these voles, most Bv109Ishowed clinical signs after short survival times(»150 d.p.i.) and were positive for PrPSc. Theremaining Bv109I and all Bv109M survivedfor longer times without showing prion-relatedpathology or detectable PrPSc. All Bv109Iwhich were previously found PrPSc-positive,


were immunonegative for pTau deposition. Incontrast, pTau deposition was detected in 16/20voles culled without clinical signs after longsurvival times (225–804 d.p.i.). pTau deposi-tion was characterized by neuropil threads andcoiled bodies in the alveus, and was similar inall voles analyzed.

These findings highlight that pTau fromGSS-F198S can propagate in voles. Impor-tantly, pTau propagation was independent fromPrPSc, as pTau was only found in PrPSc-nega-tive voles surviving longer than 225 d.p.i.Thus, selective transmission of PrPSc and pTauproteinopathies from GSS-F198S can beaccomplished by experimental transmission involes.

P.84: Transmission of chronic wastingdisease allotypes into mice

expressing elk PRNP

Jeffrey Narayan, Camilo Duque Velasquez,Chiye Kim, Nathalie Daude, Judd Aiken,

and Debbie McKenzie


Chronic Wasting Disease (CWD) is naturalprion disease affecting cervids. Polymorphismsin the cervid PRNP alleles influence host sus-ceptibility and the properties of CWD agents.This study explores the cross-species transmis-sion of CWD prions derived from white-taileddeer expressing 4 different PRNP genotypes,(Wt/Wt, Wt/H95, Wt/S96, H95/S96) into trans-genic mice expressing PrPC from Rocky Moun-tain elk. Upon onset of clinical disease, sagittalsections of brain were taken for histologicaland biochemical evaluation. As expected, therewas no transmission barrier present to TgElkmice inoculated with elk CWD, the mice pre-sented disease earlier than those with the deerisolates, at 105–110 d post inoculation (dpi).TgElk mice infected with the white-tailed deerCWD isolates presented with similar incuba-tion periods, ranging from 101–125 dpi. Theexception was the H95/S95 isolate which had asignificantly longer incubation period of 140–174 dpi. Brain sections from TgElk infected

with elk CWD agent, immunostained withBAR224, revealed intense staining with wide-spread PrPCWD distribution. The cerebrum, hip-pocampus and brainstem are all affected by theelk CWD. In contrast, PrPCWD deposition inthe TgElk infected with white-tailed deer iso-lates is less extensive and more regionalized.Further histological, biochemical and bioassayis underway to characterize the CWD agentspropagated in TgElk mice.

P.85: Improving Creutzfeldt-Jakobdisease incidence estimates by

incorporating results ofneuropathological analyses, United

States, 2003–2011

Ryan Maddox1, Marissa Person1,Arialdi Minino2, Janis Blevins3,Lawrence Schonberger1, and

Ermias Belay1

1National Center for Emerging and Zoonotic

Infectious Diseases; Centers for Disease Control

and Prevention, Atlanta GA USA; 2National Center

for Health Statistics; Centers for Disease Control

and Prevention; Hyattsville, MD USA; 3National

Prion Disease Pathology Surveillance Center; Case

Western Reserve University; Cleveland, OH USA

Introduction. The incidence of invariablyfatal prion diseases such as Creutzfeldt-Jakobdisease (CJD) can be estimated by analyzingdeath certificate data, but there are limitations.

Methods. Prion disease decedents were iden-tified from the US national multiple cause-of-death data and the National Prion DiseasePathology Surveillance Center (NPDPSC) data-base for 2003–2011. Due to limited personalidentifying information, an algorithm was con-structed to determine likely decedent matchesbetween the 2 databases. NPDPSC decedentswith a positive prion disease autopsy or biopsyresult or genetic mutation for whom no matchwas found in the multiple cause-of-death datawere added as cases for incidence calculations;those with negative neuropathology results but


with a death certificate indicating prion diseasewere removed. The resulting average annualage-adjusted incidence was then calculated.

Results. A total of 2986 decedents were iden-tified as having prion disease indicated as acause of death in the multiple cause-of-deathdata; 469 additional NPDPSC decedents wereidentified with positive neuropathology and/orgenetic findings, while 140 decedents withdeath certificates indicating prion disease hadnegative neuropathology results. Incorporatingthe matched data, the average annual age-adjusted incidence of CJD in the United Stateswas 1.2 per million.

Conclusion. Analysis of multiple cause-of-death data is an efficient means of conductingCJD surveillance. However, not all decedentsare captured as the death certificate may not listthe diagnosis; conversely, a CJD diagnosis onthe certificate may be contradicted by neuropa-thology results. Incorporating findings fromNPDPSC neuropathological and genetic analy-ses produces an estimate closer to the true inci-dence of the disease.

P.86: Estimating the risk of transmissionof BSE and scrapie to ruminants andhumans by protein misfolding cyclic

amplification

Morikazu Imamura, Naoko Tabeta,Yoshifumi Iwamaru, and

Yuichi Murayama

National Institute of Animal Health;

Tsukuba, Japan

To assess the risk of the transmission ofruminant prions to ruminants and humans atthe molecular level, we investigated the abilityof abnormal prion protein (PrPSc) of typicaland atypical BSEs (L-type and H-type) and typ-ical scrapie to convert normal prion protein(PrPC) from bovine, ovine, and human to pro-teinase K-resistant PrPSc-like form (PrPres)using serial protein misfolding cyclic amplifi-cation (PMCA).

Six rounds of serial PMCA was performedusing 10% brain homogenates from transgenicmice expressing bovine, ovine or human PrPC

in combination with PrPSc seed from typicaland atypical BSE- or typical scrapie-infectedbrain homogenates from native host species. Inthe conventional PMCA, the conversion ofPrPC to PrPres was observed only when the spe-cies of PrPC source and PrPSc seed matched.However, in the PMCA with supplements (dig-itonin, synthetic polyA and heparin), bothbovine and ovine PrPC were converted by PrPSc

from all tested prion strains. On the other hand,human PrPC was converted by PrPSc from typi-cal and H-type BSE in this PMCA condition.Although these results were not compatiblewith the previous reports describing the lack oftransmissibility of H-type BSE to ovine andhuman transgenic mice, our findings suggestthat possible transmission risk of H-type BSEto sheep and human. Bioassay will be requiredto determine whether the PMCA products areinfectious to these animals.

P.87: Mapping N-terminal/C-terminalinter-domain interactions in PrPCthrough chemical crosslinking and

tandem mass spectrometry

Alex McDonald, Deborah Leon,Christian Heckendorf, and David Harris

Boston University; Boston, MA USA

The normal physiological function of PrPC,the cellular form of PrP, and what role it playsin prion diseases remain elusive. Several linesof evidence suggest that the flexible, N-termi-nal domain (NTD) of PrPC may be responsiblefor certain toxic activities. For example, wehave shown a 21 amino acid deletion within theN-terminal domain induces spontaneous neuro-degeneration in transgenic mice and ionic cur-rents in cultured cells, with the polybasicregion (residues 23–31) being essential forthese effects. Several ligands are known to bindto the NTD, among them the divalent metalions copper (II) and zinc(II), also suggesting acritical biological function for this region.


NMR studies have previously revealed metalion-driven structural changes in recombinantPrP. Upon chelation of Cu2C or Zn2C by theoctarepeat region, the NTD transiently dockswith the CTD. Taken together, these observa-tions suggest that intramolecular interactionsbetween the NTD and CTD may play a role inregulating the physiological functions of PrPC.To further investigate this hypothesis, we haveemployed chemical crosslinking to captureNTD/CTD docked states, followed by analysisby tandem mass spectrometry to identify cross-linked residues. Preliminary evidence suggeststhat the presence of Cu2C induces increasedcross-linking between residues in the NTD andthose in the CTD. Further studies are underwayusing a variety of constructs and crosslinkers.

P.88: Prion infection interferes with rab7membrane attachment and lysosomal

degradation

Su Yeon Shim, Srinivasarao Karri, andSabine Gilch

University of Calgary; Calgary, Canada

The prion protein PrPc and its pathogenicisoform PrPSc are found at the plasma mem-brane and in the endocytic pathway of prion-infected neuronal cells. The presence of proteinaggregates attached to membranes by a glyco-syl-phosphatidyl-inositol (GPI-) anchor andprevious findings that prion-infected neuronsharbor elevated cholesterol levels let us hypoth-esize that endocytic vesicle trafficking isimpeded upon prion infection.

To verify this hypothesis we analysed themembrane association of relevant rab proteinsin N2a (mouse neuroblastoma) and ScN2a cells,respectively. Rab proteins are small GTPasesimportant for intracellular vesicle movementand targeting. They shuttle between an inactivecytosolic state and an active membrane-boundstate. Membrane association is enabled by pre-nylation of the proteins at the C-terminus. Inthe cytosol, they interact with rab GDP dissocia-tion inhibitor (rabGDI) which renders them sol-uble. When we compared the extractability of

rab7, 9 and 11 from membrane preparations ofN2a and ScN2a cells incubated with recombi-nantly expressed rabGDI, no difference wasfound. However, the overall amount of rab7associated with membranes was significantlyreduced in ScN2a. Rab7 is critical for late endo-some to lysosome maturation, and as a conse-quence of reduced active rab7 we observed aprolongated half-life of epidermal growth factorreceptor (EGFR) in ScN2a cells. When weoverexpressed the protein NPC1 which enablescholesterol efflux from late endosomes, PrPSc

propagation was significantly increased.Our data demonstrate that prion infected

neuronal cells harbor less active, membrane-associated rab7 levels. As a consequence, lyso-somal degradation is inhibited which can resultin enhanced PrPSc accumulation.

P.89: Transcriptomic determinants ofscrapie prion permissiveness in cultured

ovine microglia

Juan Mun~oz-Gutie�rrez1,Sebasti�an Aguilar-Pierl�e1,

David Schneider1,2, Timothy Baszler1, andJames Stanton3

1Department of Microbiology and Pathology;

College of Veterinary Medicine; Washington State

University; Pullman, WA USA; 2United States

Department of Agriculture; Agricultural Research

Service; Pullman, WA USA; 3Department of

Pathology; College of Veterinary Medicine;

University of Georgia; Athens, GA USA

Introduction. In cultured cells, prion permis-siveness is highly dependent on the amino acidsequence and host cellular expression of PrPC;however, PrPC expression alone is insufficient.Thus, additional factors must influence suscep-tibility to prion infection. To identify whichcellular factors are associated with permissive-ness and resistance to scrapie prions, we com-pared the transcriptional profiles of apermissive ovine microglia clone to that of anon-permissive ovine microglia clone usingRNA-Seq.


Materials and Methods. Microglia cloneswith differential prion permissiveness wereinoculated with either scrapie-positive or scra-pie-negative sheep brainstem homogenates.Prion infection was determined by ELISA andimmunoblotting. Five passages post-inocula-tion, the transcriptional profiles of microgliaclones were sequenced using Illumina technol-ogy. Raw data were mapped against the domes-tic sheep reference genome prior tocomparative transcriptional analysis.

Results. Twenty-two genes were differen-tially transcribed. In prion-resistant microglia,genes encoding for selenoprotein P, endolyso-somal proteases, and proteins involved in extra-cellular matrix remodeling, and others weresignificantly up-regulated (P < 0.05). Genesencoding for transforming growth factorb-induced, retinoic acid receptor responder 1,and phosphoserine aminotransferase 1 were up-regulated in prion-permissive microglia (P <

0.05). Gene Set Enrichment Analyses identifiedtranslation and proteolysis as the most affectedpathways.

Conclusions. In prion-resistant microglia,selenoprotein P, endolysosomal proteases, andproteins associated with extracellular matrixremodeling may act in synchrony to inhibitprion aggregation and replication. Prion-per-missiveness in ovine microglia may be favoredby the amyloid aggregating properties of thetransforming growth factor b-induced. Func-tional studies, however, are necessary to testthese associations for causality.

P.90: Flow cytometric detection of PrPSc

in neurons from prion-infected mousebrain

Takeshi Yamasaki, Akio Suzuki,Rie Hasebe, and Motohiro Horiuchi

Hokkaido University; Sapporo, Japan

Generation of an abnormal isoform of prionprotein (PrPSc) in neurons plays the central rolein a progression of neurodegeneration in prion

diseases. However, the mechanism of neurode-generation has not been fully understood.Detailed analyses of PrPSc-positive neurons inthe brain are required to clarify pathologicalchanges that occur in prion-infected neurons.Here, we report the establishment of a novelmethod for the detection of PrPSc in cells byflow cytometry with the specific labeling ofPrPSc using anti-PrP mAb 132. Neuro2a cellswere harvested by treatment with collagenaseand fixed with paraformaldehyde. After thetreatment with GdnSCN, the cells were stainedwith mAb 132 for PrPSc and analyzed by flowcytometer. The mean fluorescence intensity ofNeuro2a cells persistently infected with 22Lprion strain was 4.2-fold higher than that ofuninfected cells, indicating that PrPSc-specificstaining with mAb 132 can be applicable toflow cytometric analysis. Next, we attempted toapply this method to neural cells that were dis-sociated from brains of mice infected withprions. After gating of the cell bodies based onnuclear staining with 7-AAD and forward andside scatter profiles, the double staining ofPrPSc and neuron-specific marker NeuN clearlydistinguished NeuN-positive neurons positivefor PrPSc from NeuN-positive neurons negativefor PrPSc. These results indicate that thismethod is applicable to the separation of PrPSc-positive neurons from mice brains by fluores-cence-activated cell sorting, and thus allows usto perform prion-infected neural cell-type spe-cific analyses using transcriptome or proteometechnique.


P.91: Prion-based regulation of thedynamic changes in ribonucleoprotein

complexes

Irina Derkatch1,*, Catherine Potenski2,Xiang Li1, and Eric Kandel1,3,4,5

1Department of Neuroscience; College of

Physicians and Surgeons of Columbia University;

New York, NY USA; 2Depts. Microbiology and

Biochemistry and Molecular Pharmacology; NYU

School of Medicine; New York, NY USA; 3New York

State Psychiatric Institute; New York, NY USA;4Kavli Institute of Brain Science; College of

Physicians and Surgeons of Columbia University;

New York, NY USA; 5Howard Hughes Medical

Institute; College of Physicians and Surgeons of

Columbia University; New York, NY USA

Prions are over-represented among RNA-binding proteins and components of RNP com-plexes regulating biogenesis, translation, turn-over and cellular distribution of mRNAs. Weprovide 2 new lines of evidence that prion-based complexes are engaged in dynamic rear-rangements in a network of RNA-processingcomplexes allowing for rapid switchingbetween RNA storage and degradation, andbetween inhibition and activation of proteinsynthesis. Analysis of prion-like aggregationdirected by the Q/N-rich prion domain of yeastLsm4, a P-body-associated activator of mRNAdecapping, revealed that Lsm4 forms heritableaggregates. The aggregation, that is controlledby chaperones and induced by environmentalchanges, such as temperature drop to 4�C, leadsto the increase in the number of P-bodies andtheir clustering around Lsm4 aggregates, indic-ative of the directed modulation of mRNA turn-over. Analysis of prion properties of yeast Pub1and its mammalian homolog Tia1 revealed thatthis protein participates in 2 distinct self-per-petuating structures, both formed through itsQ/N-rich prion domain. One is localized to P-bodies and stress granules, consistent withknown role of Pub1/Tia1 in stress granuleassembly. The other structure is formed co-operatively by Pub1/Tia1 and Sup35/Gspt2, theeRF3 release factor. This heteroprotein prion isnormally present in yeast cells, can be

visualized as lines forming along tubulin cyto-skeleton and drives the assembly of an RNPcomplex implicated in maintaining the integrityof microtubule cytoskeleton. We hypothesizethat the complex directs tubulin synthesis to thesites of microtubule assembly, and that Pub1/Tia1 functionally shuffles between 2 prion-likestructures. Support: NIH grant7R01GM070934–06 (ILD), HHMI (ERK).

P.92: The prion protein inchemosensitive cells of the carotid bodyin uninfected and infected hamsters

Anthony Kincaid, Melissa Clouse,Albert Lorenzo, and Jason Bartz

Creighton University; Omaha, NE USA

The carotid bodies (CB) are highly vascular-ized mammalian sensory organs consisting ofuniquely organized clusters of chemosensitivecells that function to monitor blood gasses andadjust respiration accordingly. The chemosen-sitive cells are synaptically linked to glosso-pharyngeal nerve terminals that project into thenucleus of the solitary tract in the medulla andto sympathetic preganglionic neurons, bothknown to be sites of early prion accumulationin experimental prion pathogenesis. We exam-ined the CBs of uninfected golden Syrian ham-sters for the presence of the normal isoform ofthe prion protein (PrPC), required for prion rep-lication and spread, to establish whether thesestructures might be involved in prion neuroin-vasion following prionemia. We also examinedthe CBs of clinically-ill hamsters intracere-brally inoculated with either the HY or DYstrain of hamster-adapted transmissible minkencephalopathy (TME) to determine if CB cellsaccumulate the disease associated form of theprion protein (PrPd) after infection. PrPC wasidentified in the chemosensitive cells in CBs ofuninfected hamsters, and PrPd was detected inthe chemosensitive cells of animals inoculatedintracerebrally with HY TME infected brainhomogenate, but not those animals inoculatedwith DY TME infected brain homogenate. Thisis the first report demonstrating that the


chemosensitive cells of the CB express PrPC

and that they are a site of disease associatedprion accumulation in clinically ill animals.The results of these studies indicate that theCBs are a possible nexus for the centripetaland/or centrifugal spread of prions betweenblood and the nervous system in infectedanimals.

P.93: Modeling genetic prion diseases intransgenic mice expressing mutant

bank vole PrP

Joel Watts1,2, Kurt Giles1,Matthew Bourkas2, Smita Patel1,Marta Gavidia1, Abby Oehler1,

Stephen DeArmond1, and Stanley Prusiner1

1Institute for Neurodegenerative Diseases;


Francisco, CA USA; 2Tanz Centre for Research in

Neurodegenerative Diseases; University of

Toronto; Toronto, ON, Canada

Unlike other rodents, bank voles exhibit anunprecedented susceptibility to prions frommany different species, a phenomenon solelydue the sequence of the bank vole prion protein(BVPrP). We recently demonstrated that trans-genic (Tg) mice expressing wild-type (wt)BVPrP containing isoleucine at polymorphiccodon 109 develop a spontaneous neurodegen-erative disorder that exhibits many of the hall-marks of prion disease, including thegeneration of prion infectivity. To determine ifmutations that cause genetic prion disease inhumans affect the manifestation of spontaneousdisease, we generated Tg mice expressingBVPrP containing either the D178N mutation,which causes fatal familial insomnia; theE200K mutation, which causes familial Creutz-feldt-Jakob disease; or an anchorless PrP muta-tion similar to those that cause Gerstmann-Str€aussler-Scheinker disease. Despite similarlevels of BVPrP DNA and mRNA in the brain,BVPrP protein levels were much lower in Tgmice expressing mutant BVPrP than in Tgmice expressing wt BVPrP, suggesting that themutations destabilize the structure of BVPrP.

Remarkably, these physiological or subphysio-logical concentrations of mutant BVPrPresulted in highly penetrant spontaneous dis-ease, with mean incubation periods rangingfrom»120 to»460 days. The brains of sponta-neously ill mice exhibited prominent prion dis-ease-specific neuropathology that was uniqueto each mutation as well as a highly proteinaseK–resistant PrP fragment. Moreover, the spon-taneously formed prions were transmissible toTg mice expressing wt BVPrP or wt mousePrP. Our results suggest that BVPrP may facili-tate the generation of superior mouse models ofinherited human prion diseases.

P.94: Increased infectivity of anchorlessmouse scrapie prions in transgenic miceoverexpressing human prion protein

Brent Race, Katie Phillips,Kimberly Meade-White, James Striebel,

and Bruce Chesebro

NIAID Rocky Mountain Laboratories; Hamilton,

MT USA

Prion protein (PrP) is found in all mammalsmostly as a glycoprotein anchored to theplasma membrane by a C-terminal glycophos-phatidylinositol (GPI) linkage. Following prioninfection, host protease-sensitive prion protein(PrPsen) is converted into an abnormal, dis-ease-associated, protease-resistant form(PrPres). Biochemical characteristics such asthe PrP amino acid sequence and post-transla-tional modifications such as glycosylation andGPI anchoring, can affect the transmissibilityof prions as well as the biochemical propertiesof the PrPres generated. Previous in vivo stud-ies have tested the roles of amino acid sequenceand glycosylation on cross-species transmis-sion, but the role of GPI anchoring has not beentested. In the current studies we examined theeffect of PrPres GPI anchoring using a mouse-human species barrier model. In this model,anchorless 22L mouse scrapie prions weremore infectious than anchored 22L mouse scra-pie prions when inoculated into tg66 transgenicmice, which expressed wild-type anchored


human PrP at 8–16 fold above normal. Thus thelack of the GPI anchor on PrPres appeared toreduce the effect of the mouse-human PrP spe-cies barrier. In contrast, neither form of 22Lprions induced disease when tested in a secondtransgenic mouse which expressed human PrPat 2–4 fold above normal suggesting that PrPexpression level also had an impact on ourmodel.

P.95: Glycosylation of PrPC is a keyfactor in determining TSE transmission

between species

Frances Wiseman1, Enrico Cancellotti1,Pedro Piccardo1, Kayleigh Iremonger1,

Aileen Boyle1, Deborah Brown1,James Ironside2, Jean Manson1, and

Abigail Diack1

1The Roslin Institute; University of Edinburgh;

Easter Bush, UK; 2The National CJD Research &

Surveillance Unit; University of Edinburgh;

Edinburgh, UK

The risk of transmission of transmissiblespongiform encephalopathies (TSE) betweendifferent species has been notoriously unpre-dictable because the mechanisms of transmis-sion are not fully understood. A transmissionbarrier between species often prevents infectionof a new host with a TSE agent. Nonetheless,some TSE agents are able to cross this barrierand infect new species with devastating conse-quences. The host PrPC misfolds during diseasepathogenesis and has a major role in controllingthe transmission of agents between species, butsequence compatibility between host and agentPrPC does not fully explain host susceptibility.PrPC is post-translationally modified by theaddition of glycan moieties which have animportant role in the infectious process. Herewe show in vivo that glycosylation of the hostPrPC has a significant impact on the transmis-sion of TSE between different host species.

We infected mice in which the first (N180T),second (N196T) or both (N180T and N196T)N-glycan attachment sites are disrupted with 2human agents (sCJDMM2 and vCJD) and one

hamster strain (263K). The absence of glyco-sylation at both or the first PrPC glycosylationsite in the host results in almost complete resis-tance to disease. Absence of the second glyco-sylation site has a dramatic effect on the barrierto transmission between host species, facilitat-ing the transmission of sCJDMM2 to a hostnormally resistant to this agent. These resultsdemonstrate that glycosylation of host PrPC candramatically alter cross species transmissionand is a key factor in determining the transmis-sion efficiency of TSEs between differentspecies.

P.96: Variable relative contribution ofmethionine and valine at residue 129 to

protease resistant prion protein inheterozygous cases of Creutzfeldt-Jakob

disease

Roger Moore1, Mark Head2,James Ironside2, Gianluigi Zanusso3,Young Pyo Choi4, and Suzette Priola1

1Rocky Mountain Laboratories; NIH; NIAID;

Hamilton, MT US; 2University of Edinburgh;

Edinburgh, UK; 3University of Verona; Verona,

Italy; 4Korea Brain Research Institute; Daegu,

Republic of Korea

Sporadic Creutzfeldt-Jakob disease (sCJD)is thought to originate from the spontaneousmisfolding of the endogenous host prion pro-tein (PrPC) into its infectious prion isoform,PrPSc. By contrast, iatrogenic CJD (iCJD) isassociated with exposure to an exogenoussource of PrPSc. sCJD and iCJD occur as 3 pos-sible PRNP codon 129 genotypes: patientshomozygous for methionine (M129) or valine(V129) and those who are heterozygous at thislocus. In CJD patients heterozygous at residue129, the relative contribution of each allotypeto PrPSc is unknown and its influence on prionpathogenesis is poorly understood. We haveused mass spectrometry to determine the rela-tive abundance of M129 and V129 in PrPScfrom heterozygous cases of sCJD and iCJD, thelatter of which are linked to human growth hor-mone therapy in the United Kingdom. Our


results show that, while the amount of M129 orV129 in PrPSc is variable in heterozygoussCJD patients, PrPSc with V129 is most abun-dant in the majority of heterozygous iCJDpatients. The relative abundance of M129 orV129 in PrPSc did not correlate with CJD type,age at clinical onset, or disease duration. How-ever, the data are consistent with sCJD PrPScoriginating from the stochastic refolding ofendogenous PrPC and iCJD originating from anon-stochastic, exogenous source of PrPSc.Thus, the relative abundance of M129 andV129 in PrPSc may be indicative of the PrPScallotype(s) which best converted PrPC to PrPScand may provide a means to trace back to theorigin of CJD infection.

P.97: Scrapie transmits to white-taileddeer by the oral route and has a

molecular profile similar to chronicwasting disease and distinct from the

scrapie inoculum

Justin Greenlee1, S Jo Moore1, Jodi Smith1,M Heather West Greenlee2, and

Robert Kunkle1

1National Animal Disease Center; Ames, IA USA;2Iowa State University; Ames, IA USA

The purpose of this work was to determinesusceptibility of white-tailed deer (WTD) tothe agent of sheep scrapie and to compare theresultant PrPSc to that of the original inoculumand chronic wasting disease (CWD). We inocu-lated WTD by a natural route of exposure (con-current oral and intranasal (IN); n D 5) with aUS scrapie isolate. All scrapie-inoculated deerhad evidence of PrPSc accumulation. PrPSc wasdetected in lymphoid tissues at preclinical timepoints, and deer necropsied after 28 monthspost-inoculation had clinical signs, spongiformencephalopathy, and widespread distribution ofPrPSc in neural and lymphoid tissues. Westernblotting (WB) revealed PrPSc with 2 distinctmolecular profiles. WB on cerebral cortex hada profile similar to the original scrapie inocu-lum, whereas WB of brainstem, cerebellum, or

lymph nodes revealed PrPSc with a higher pro-file resembling CWD. Homogenates with the 2distinct profiles from WTD with clinical scra-pie were further passaged to mice expressingcervid prion protein and intranasally to sheepand WTD. In cervidized mice, the 2 inoculahave distinct incubation times. Sheep inocu-lated intranasally with WTD derived scrapiedeveloped disease, but only after inoculationwith the inoculum that had a scrapie-like pro-file. The WTD study is ongoing, but deer inboth inoculation groups are positive for PrPSc

by rectal mucosal biopsy. In summary, thiswork demonstrates that WTD are susceptible tothe agent of scrapie, 2 distinct molecular pro-files of PrPSc are present in the tissues ofaffected deer, and inoculum of either profilereadily passes to deer.

P.98: Super-infection of knock-in mousemodels of familial prion diseases revealsdifferences in selective vulnerability

Walker Jackson

German Center for Neurodegenerative Diseases;

Bonn, Germany

Why do neurodegenerative diseases tend totarget specific brain regions? To begin toaddress this phenomenon known as selectivevulnerability we combined two powerful tools,an allelic series of Prnp knock-in mouse lineswith the high precision of mouse adapted scra-pie strains. The first mouse line modeled fatalfamilial insomnia (FFI), and in old age sponta-neously developed neuronal loss and reactivegliosis in the thalamus. The second line mod-eled Creutzfeldt-Jakob disease (CJD), spontane-ously developing PrPres aggregates, spongiosis,and reactive gliosis in the hippocampus. In bothmodels the cerebellum was also affected: atro-phied in the FFI model and loaded with PrPres

aggregates in the CJD model. A third mouseline expressed a mutated N-terminal polybasicregion (PBR), but was disease free. We there-fore challenged our allelic series with two scra-pie strains with similar incubation periods(RML and 22L), one of which was reported to


aggressively target the cerebellum in vivo(22L). Based on incubation periods, the FFImice were highly resistant to both scrapiestrains. In contrast, CJD mice were resistant to22L but very sensitive to RML. The oppositewas true for PBR mice which were highly resis-tant to RML but much more prone to 22L thanFFI and CJD mice. Interestingly, multiplemarkers of neurodegeneration revealed a diver-gence of responses to the two strains in variousbrain regions. In particular, various markers ofgliosis appeared in different brain regionsdepending on the mouse line:scrapie strain com-bination, suggesting the existence of a diversityof glial phenotypes in these brains.

P.99: Preliminary study of infectivity inperipheral tissues from a transgenicmodel of spontaneous prion disease

Laura Pirisinu1, Michele Angelo Di Bari1,Claudia D’Agostino1, Stefano Marcon1,Geraldina Riccardi1, Paolo Frassanito1,

Natalia Fern�andez-Borges2,Manuel S�anchez-Mart�ın3,

Joaquin Castilla1,4, Umberto Agrimi1, andRomolo Nonno1

1Department of Veterinary Public Health and Food

Safety; Istituto Superiore di Sanit�a; Rome, Italy;2CIC bioGUNE; Parque tecnol�ogico de Bizkaia;Derio, Spain; 3Unidad de generaci�on de OMGs.

S.E.A. Dpt. of Medicine; University Salamanca;

Salamanca, Spain; 4IKERBASQUE; Basque

Foundation for Science; Bilbao, Spain

Transgenic mice over-expressing bank voleprion protein (TgL1), carrying isoleucine atPrP codon 109 (BvPrP109I), were reported todevelop a spontaneous neurological diseaserecapitulating the hallmarks of prion diseases.Furthermore, we detected infectivity in thebrain of spontaneously sick TgL1, by bioassayin wild type voles (Bv109I), up to 10¡8 braindilution. In this study we investigated the distri-bution of infectivity in peripheral tissues (skel-etal muscles, spleen and blood) from terminallysick TgL1 mice by bioassay in wild type voles

Bv109I. All inocula coming from skeletal mus-cle (n D 3) were infectious, resulting in 80–100% attack rate with mean survival times of127, 145 and 199 dpi. By comparison withbrain end point dilution experiments in the wildtype vole model, the infectivity in muscles canbe estimated to be between 102 and 105 foldless than in brains. In contrast, spleen derivedinocula (n D 4) didn’t induce disease in anywild type vole Bv109I. Whole blood inocula(nD 2) transmitted the disease in only 1/23 ani-mals at 375 dpi.

The absence of infectivity in spleen and thepresence of infectivity traces in blood couldsuggest that the LRS doesn’t play a major rolein the spontaneous disease in TgL1 mice. Incontrast, TgL1 mice produce high levels ofinfectivity in skeletal muscles other than inCNS. Further investigations might help tounderstand the kinetics and pathophysiologicalevents underlying accumulation of PrPSc andprion infectivity in muscle, and which celltype/s and functional structure/s are involved inaffected muscles.

P.100: Cyclophilin A deficiencyexacerbates RML-induced prion disease

Liliana Comerio, Ihssane Bouybayoune,Laura Pasetto, Valentina Bonetto, and

Roberto Chiesa

IRCCS - Mario Negri Institute for Pharmacological

Research; Milano, Italy

Prion diseases are typically characterized bydeposition of abnormally folded, partially prote-ase-resistant prion protein, which is associatedwith activated glia and increased release of cyto-kines. This neuroinflammatory response mayplay a role in disease pathogenesis. Recentstudies indicated that cyclophilin A (CyPA) maybe a key mediator of the neuroinflammatoryresponse in prion diseases. It was found thatCyPA from scrapie-infected brains inducedcytokine release from astroglia and microglia invitro. This effect was reduced by both anti-CyPAantibody and cyclosporine A, a CyPA inhibitor


(Tribouillard-Tanvier et al., 2012). However, therole of CyPA in vivowas not studied.

To investigate whether CyPA mediates neu-roinflammation and influences prion diseasepathogenesis, we inoculated CyPAC/C, CyPAC/

¡ and CyPA¡/¡ mice with the RML scrapiestrain, and monitored the time to onset of neu-rological signs of disease and survival. Time toonset of disease in CyPA¡/¡ mice was reducedby 3% and 6% compared to CyPAC/¡ andCyPAC/C mice, respectively (p < 0.05). Timeto death was also significantly reduced, withCyPA¡/¡ mice surviving 3.5% and 7% lessthen CyPAC/¡and CyPAC/C mice (p < 0.05and p < 0.001, respectively). Thus, CyPA defi-ciency exacerbated RML-induced disease.Analysis of glial activation is in progress toestablish the contribution of CyPA-mediatedneuroinflammation in disease pathogenesis.

Reference

[1] Tribouillard-Tanvier D, Carroll JA, Moore RA, Strie-

bel JF, Chesebro B. Role of cyclophilin A from brains

of prion-infected mice in stimulation of cytokine

release by microglia and astroglia in vitro. J Biol

Chem 2012; 287: 4628–39; PMID:22179611

P.101: Complement receptors andregulatory proteins directly bind prions

and assist in pathogenesis

Sarah Kane, Taylor Farley, and Mark Zabel


Complement, a component of the innateimmune system, is crucial for establishingprion disease. The work in this study aimed tobiochemically characterize the interactionsbetween Complement and prions, as well asdetermine the role of a Complement regulatoryprotein in establishing disease. We employedsurface plasmon resonance (SPR) to test inter-actions between a panel of Complement pro-teins and enriched Chronic Wasting Diseaseprion rods. Upon identification of CD21/35 asputative prion receptors by SPR, we injectedfluorescent rods into the footpad and performedintravital microscopy of the popliteal lymph to

test for co-localization in vivo. Our findingssuggest Complement Receptors 1/2 (CD21/35)are putative prion receptors because not onlydid we observe an interaction between CD21/35 and prions via SPR, fluorescent prions co-localized with CD21/35 in vivo on B and follic-ular dendritic cells in the popliteal lymph nodewithin an hour of inoculation. We also deter-mined other Complement proteins, includingC3 cleavage products and C1q, bound infec-tious prions using SPR. Lastly, to determine therole of complement regulatory protein factor Hin disease pathogenesis, we tested a gene-doseeffect in response to mouse-adapted Scrapieprions. Factor H deficient mice survived longerand accumulated less prions than their hemizy-gous or wild type littermates. These findingsidentify CD21/35 as prion receptors and, assuch, provide viable targets for therapeuticintervention. Preventing either or both CD21/35 and factor H from interacting with prionscould delay or even prevent disease.

P.102: Increased membranepermeability by complement factorsaffect PrPSc deposition in neurons

Rie Hasebe, Akio Suzuki,Takeshi Yamasaki, and Motohiro Horiuchi

Graduate School of Veterinary Medicine; Hokkaido

University; Sapporo, Japan

We previously reported that reaction of com-plement factors induced translocation of phos-phatidylserine on the plasma membrane ofscrapie-infected N2a cells (Hasebe et al., Virol-ogy, 2012). We assessed here if complementfactors induces similar consequences in pri-mary-cultured cortical neurons from mousefetuses. When neurons were infected with theChandler and 22L strains, PrPSc was detectedby immunoblotting after 10 days post inocula-tion (dpi). We used normal mouse serum(NMS) as a source of complement factors.NMS treatment at 17 dpi increased uptake ofpropidium iodide (PI) in the prion-infected neu-rons time-dependently. However, PI-uptake inthe neurons at 24 dpi was largest at 6 h after


the treatment and reduced thereafter. No mor-phological change was observed NMS-treatedneurons. These results suggest that increased PIuptake by NMS treatment resulted from tempo-rarily increased membrane permeability ratherthan cell death. Next, we examined if theincreased membrane permeability influencesdeposition of PrPSc in the neurons. NMS treat-ment at 20 dpi reduced the amount of PrPSc inthe Chandler-infected neurons at 8 days aftertreatment. On the other hand, the amount ofPrPSc in the 22L-infected neurons increased byNMS treatment at 12 dpi but not at 20 dpi. Thechange in the amount of PrPSc was blocked bypretreatment of NMS with anti-complement anti-bodies. These results suggest that complementfactors increased membrane permeability onprion-infected neurons, but the effects on PrPSc

deposition were different among prion strains.

P.103: A novel insertion mutation inPrnp causes Gerstmann-Str€aussler-Scheinker Disease in transgenic mice

Robert Mercer1,2, Charles Mays1,Hristina Gapeshina1, Lyudmyla Dorosh3,4,Serene Wohlgemuth1, Nathalie Daude1,Jing Yang1, Kerry Ko1, Holger Wille1,5,Neil Cashman6, Michael Coulthart7,

Maria Stepanova3,4, andDavid Westaway1,2


University of Alberta; Edmonton, Alberta, Canada;2Department of Medicine; University of Alberta;

Edmonton, Alberta, Canada; 3National Research

Council of Canada; Edmonton, Alberta, Canada;4Department of Electrical and Computer

Engineering; University of Alberta; Edmonton,

Alberta, Canada; 5Department of Biochemistry;

University of Alberta; Edmonton, Alberta, Canada;6Division of Neurology; University of British

Columbia; Vancouver, British Columbia, Canada;7Canadian Creutzfeldt-Jakob Disease Surveillance

System; Public Health Agency of Canada;

Winnipeg, Manitoba, Canada

Recently, the Canadian CJD SurveillanceSystem discovered a novel 24 base pair

insertion mutation of PRNP in a Gerstmann-Str€aussler-Scheinker Disease patient (Hinnellet al. 2011). This mutation is predicted toextend the length of the hydrophobic domain(HD) by 8 residues which is of great interest inPrPC biology because: i) the HD is the mosthighly conserved segment of the protein, ii) ithas been implicated in many of the proposedfunctions of PrPC and iii) it is thought to beinvolved in the early structural rearrangementsduring the transition between PrPC and PrPSc.We created transgenic mice expressing thismutated allele within the context of murine PrPand determined that these animals develop aspontaneous neurologic syndrome at ages>300 d. The onset of disease in these mice canbe accelerated through intracranial inoculationwith brain homogenate from clinically ill ani-mals, demonstrating transmissibility. Histo-pathological analysis of these mice showsvacuolation and prominent gliosis. Biochemicalprofiling reveals a 7 kDa PrP fragment follow-ing exposure to proteinase K or removal of car-bohydrates, a characteristic of GSS prions.Molecular dynamics simulations indicate anincrease in the proportion of b-sheet content,which may prove useful in deciphering theearly structural changes during the process ofprion conversion.

P.104: Neuroprotective role of innateimmunity in prion infection

Sang-Gyun Kang, Chiye Kim,Leonardo M Cortez, Jing Yang,

Mar�ıa Carmen Garza, Holger Wille,Valerie L Sim, David Westaway,Debbie McKenzie, and Judd Aiken

Centre for Prions and Protein Folding Diseases;

University of Alberta; Edmonton, Alberta, Canada

Prion diseases are characterized by the self-aggregation of abnormally folded prion proteininto amyloid. This amyloidosis is associatedwith synaptic and neuronal loss, vacuolationand neuroinflammation in the central nervoussystem (CNS). Astrocytes, the most abundantimmune competent cells in the CNS,


participate in the local innate immuneresponses triggered by a variety of insultsincluding amyloidogenic proteins such as prionprotein, amyloid-b and a-synuclein. It is notclear whether innate immune molecules andcells have neurotoxic or neuroprotective rolesin neurodegenerative diseases. In this study,astrocytes in culture were permissive to prioninfection under certain conditions. Pretreatmentof primary cerebellar granule neurons andastrocytes culture with lipopolysaccharide(LPS) resulted in a dramatic reduction in prionreplication compared to non-treated control asdetermined by Western blot, immunocyto-chemistry and animal bioassay. Suppression ofLPS-induced immune responses in astrocytes,however, increased prion replication. Astro-cytes triggered up-regulation of Toll-like recep-tors and production of cytokines in response torecombinant prion fibrils. Our results suggestthat astrocytes may play a neuroprotective rolein the early stage of prion infection.

P.105: RT-QuIC models trans-speciesprion transmission

Kristen Davenport, Davin Henderson,Candace Mathiason, and Edward Hoover

Prion Research Center; Colorado State University;

Fort Collins, CO USA

The propensity for trans-species prion trans-mission is related to the structural characteris-tics of the enciphering and heterologous PrP,but the exact mechanism remains mostly mys-terious. Studies of the effects of primary or ter-tiary prion protein structures on trans-speciesprion transmission have relied primarily uponanimal bioassays, making the influence of prionprotein structure vs. host co-factors (e.g. cellu-lar constituents, trafficking, and innate immuneinteractions) difficult to dissect. As an alterna-tive strategy, we used real-time quaking-induced conversion (RT-QuIC) to investigatetrans-species prion conversion.

To assess trans-species conversion in theRT-QuIC system, we compared chronic wast-ing disease (CWD) and bovine spongiform

encephalopathy (BSE) prions, as well as felineCWD (fCWD) and feline spongiform encepha-lopathy (FSE). Each prion was seeded intoeach host recombinant PrP (full-length rPrP ofwhite-tailed deer, bovine or feline). We demon-strated that fCWD is a more efficient seed forfeline rPrP than for white-tailed deer rPrP,which suggests adaptation to the new host.Conversely, FSE maintained sufficient BSEcharacteristics to more efficiently convertbovine rPrP than feline rPrP. Additionally,human rPrP was competent for conversion byCWD and fCWD. This insinuates that, at thelevel of protein:protein interactions, the barrierpreventing transmission of CWD to humans isless robust than previously estimated.

P.106: Recovery of agents with ‘slowvirus’ properties from prion positive

sheep with scrapie

William Todd

Retired professor; Department of Pathobiological

Sciences; Louisiana State University; Baton Rouge,

LA USA

The prion theory of ‘proteinaceous infec-tious particles’ was proposed after interpretingdata in a manner that excluded pathogens as thecause for scrapie, a naturally transmitted neuro-degenerative disease of sheep. Prions wereoffered as foreign entities, but they are hostproteins modified in prion disease. Becausehost prions, modified as PrPres, resist biocides,cause spongiform encephalopathy and convertPrPC into PrPres, they transfer prion diseasepathology by injection into brains, even if braintissues containing PrPres were biocide pre-treated. When sheep are diagnosed with scrapiemany of their PrPC had been converted intoPrPres, the cause of scrapie signs and pathology.What caused this conversion? Because patho-gens can modify host proteins as pathogenicmechanisms we attempted to culture putativepathogens from sheep with scrapie whileexcluding the prion pathogenic mechanism.From four prion positive sheep, agents contain-ing nucleic acids were recovered. These agents


express two distinct modes of function. Withinhost cells their virus-like expression producesvirus like particles comparable to those pub-lished (Bignami and Parry, 1971 Science 171:389–390. Payne and Sibley, 1975 Acta neuro-pathology 31: 353–361. Manuelidis et al., 2007PNAS 104: 1965–1970). Their prokaryotemode of gene function seems only to occur out-side host cells and outside the CNS. Thoseproducts are tough structures likely for genomepreservation such as seeding fields. These scra-pie agents are proposed as hybrid pathogensand are available from the NIAID Repositoryfor Biosecurity and Emerging Infectious Dis-eases, deposited by LSU.

P.107: Inhibition of the interactionbetween amyliod-b and prion protein by

computational methods: A strategyagainst Alzheimer’s disease

Michael Kontaiteh Fonjang1,2,3,4

1Ndowecam Entreprise; Yaounde, Cameroon;2International School of Advanced Studies; Trieste,

Italy; 3Lund University; Lund, Sweden; 4University

of Buea; Buea, Cameroon

The inhibition of the interaction between theprion and amyloid-b is a valuable strategyagainst Alzheimer’s disease. However the com-pounds that bind the N-term_HuPrP such as theporphyrins have poor blood brain barrier per-meability. Compounds similar to the porphyr-ins, with good blood-brain barrier permeabilitymight be ideal drug candidates againstAlzheimer’s disease. In order to select the por-phyrin-like compounds from the ZINC data-base, we imputed the porphyrin SMILE or itsname, and obtained 36 porphyrin-like mole-cules. From the 36, we selected 29 of them withlipophilicity coefficients in the range (0, 5), andmolecular weight less than 500. However, wetolerated some lipophilicity coefficients thanproscribed by the Lipinski’s rule of 5 becauseof the uncertainties in the calculated values. Tocalculate logP or logS coefficient using theMolinspiration algorithm or the AlOGP2.1, wecopied the SMILE or draw the structure of theligand from the ZINC database, imputed it on

the platform, and then click on calculate LogPor LogS coefficient. The calculation is per-formed in a matter of seconds, and the resultsdisplayed. The minimum value for LogP being¡0.4 for ZINC59380156 and the maximum is4.55 for ZINC78206861. The ZINC databaseuses the fragmental method of Molinspirationin the calculation of the Lipophilicity coeffi-cient wich takes into account correction factorsthat compensate for intermolecular interrac-tions; consequently we suggest the 29 com-pounds selected from the ZINC database willbe used for molecular docking with N-Term_PrP, in order to select the best bindingposes for molecular dynamics simulations.

P.108: Successful oral challenge of adultcattle with classical BSE

Sandor Dudas1,*, KristinaSantiago-Mateo1, Tammy Pickles1,

Catherine Graham2, and Stefanie Czub1

1Canadian Food Inspection Agency; NCAD

Lethbridge; Lethbridge, Alberta, Canada; 2Nova

Scotia Department of Agriculture; Pathology

Laboratory; Truro, Nova Scotia, Canada

Classical Bovine spongiform encephalopa-thy (C-type BSE) is a feed- and food-bornefatal neurological disease which can be orallytransmitted to cattle and humans. Due to thepresence of contaminated milk replacer, it isgenerally assumed that cattle become infectedearly in life as calves and then succumb to dis-ease as adults.

Here we challenged three 14 months old cat-tle per-orally with 100 grams of C-type BSEbrain to investigate age-related susceptibility orresistance. During incubation, the animals weresampled monthly for blood and feces and sub-jected to standardized testing to identifychanges related to neurological disease.

At 53 months post exposure, progressivesigns of central nervous system disease wereobserved in these 3 animals, and they wereeuthanized. Two of the C-BSE animals testedstrongly positive using standard BSE rapidtests, however in 1 C-type challenged animal,


PrPsc was not detected using rapid tests forBSE. Subsequent testing resulted in the detec-tion of pathologic lesion in unusual brain loca-tion and PrPsc detection by PMCA only.

Our study demonstrates susceptibility ofadult cattle to oral transmission of classicalBSE. We are further examining explanationsfor the unusual disease presentation in the thirdchallenged animal.

P.109: Development andcharacterization of an ex-vivo brain sliceculture model of chronic wasting disease

Naveen Kondru1, Justin Greenlee2,Heather Greenlee1, Sireesha Manne1,Qingzhong Kong3, Patrick Halbur4,

Arthi Kanthasamy1, andAnumantha Kanthasamy1

1Department of Biomedical Sciences; College of

Veterinary Medicine; Iowa State University; Ames,

IA USA; 2Virus and Prion Research Unit; National

Animal Disease Center; Agricultural Research

Service; United States Department of Agriculture;

Ames, IA USA; 3Departments of Pathology and

Neurology; Case Western Reserve University;

Cleveland, OH USA; 4Veterinary Diagnostic and

Production Animal Medicine; College of Veterinary

Medicine; Iowa State University; Ames, IA USA

Prion diseases have long incubation timesin vivo, therefore, modeling the diseases ex-vivo will advance the development of rationale-based therapeutic strategies. An organotypicslice culture assay (POSCA) was recentlydeveloped for scrapie prions by inoculatingmouse cerebellar brain slices with prions. How-ever, efforts to develop a POSCA model forchronic wasting disease (CWD) have failed dueto the species barrier between mice and cervids.To overcome this difficulty, we adopted a trans-genic cervidized (Tg12) mouse model and suc-cessfully developed an ex-vivo brain sliceculture model for CWD. We incubated 350-mmcerebellar slices from Tg12 mouse pups withCWD brain homogenate and washed themthoroughly. With the genotypes of the pupsblinded, the cultures were grown over

42–48 days before being tested for CWD infec-tivity using the recently developed RT-QuICassay. Slices from Tg12 cervidized pups withPrPC/¡ genotype tested positive but slicesfrom the Tg12 PrP¡/¡ genotype did not.Infectivity was present in 11 out of 11 Tg 12PrPC/¡, whereas 0 out of 10 Tg12 PrP¡/¡.We have successfully cultured POSCA brainslices infected with RML scrapie as confirmedby RT-QuIC and PK digestion assays. Bio-chemical studies revealed degenerative changesand oxidative damage in the prion infected slicecultures. Our results demonstrate that combin-ing the brain slice culture model of prion dis-eases with the RT-QuIC assay offers apromising platform for studying the mecha-nisms of prion proteinopathies as well as forscreening anti-prion therapeutics. (ISU Presi-dential Wildlife initiative, ISU-CVM Diagnos-tic lab and NIH ES19267 and NS074443).

P.110: Prion protein gene sequencesanalysis in twelve sheep breeds

of Pakistan

Mohammad Farooque Hassan*

China Agricutural University; Beijing, China

Prions are considered the only agents ofTransmissible Spongiform Encephalopathies(TSEs) and are harmful pathogens of mammals.These infectious agents of host are made upthrough aggregation of conformational isomers(PrPSc) and encode glycoprotein (PrPC) of33–35 kDa. TSEs are fatal group of diseaseswhich are neurodegenerative and includechronic wasting disease in deer and elk, Creutz-feldt-Jakob disease (CJD) and transmissiblemink encephalopathy (TME) in humans andscrapie in goats and sheep. The accumulationof abnormal form of the normal protein (PrP) iscommon in all diseases related TSE. Thisabnormal form of PrP called PrPSc is resistantto proteolysis as well as infectious. Presentstudy was conducted in order to do sequenceanalysis of prion protein gene in 12 breeds (n D129) of the sheep from all provinces of Pakistanincluding Azad Jammu and Kashmir. We


amplified 771 bp of PrP gene in selected 12sheep breeds followed by sequencing. We iden-tified single nucleotide polymorphisms (SNPs)and some heterozygous sites were detected inaligned sequences using CodonCode Aligner.We compared the results with other sheepbreeds of the word and reported mammalianspecies sequences in GenBank NCBI throughUPGMA phylogenetic analysis using MEGA6.1. This study provided useful PrP gene basedinformation in sheep population across thecountry.

P.111: W8, a new Sup35 prion strain,transmits distinctive information with a

conserved assembly scheme

Yu-Wen Huang1,2, Yuan-Chih Chang3,Ruben Diaz-Avalos4, and Chih-Yen King2

1Molecular and Cell Biology; Taiwan International

Graduate Program; Academia Sinica and National

Defense Medical Center; Taipei, Taiwan; 2Institute

of Molecular Biology; Academia Sinica; Taipei,

Taiwan; 3Institute of Cellular and Organismic

Biology; Academia Sinica; Taipei, Taiwan; 4Janelia

Farm Research Campus; Howard Hughes Medical

Institute; Ashburn VA USA

Prion strains are different self-propagatingconformers of the same infectious protein.Three strains of the [PSI] prion, infectiousforms of the yeast Sup35 protein, have beenpreviously characterized in our laboratory.Here we report the discovery of a new [PSI]strain, named W8. We demonstrate its robustcellular propagation as well as the protein-onlytransmission. To reveal strain-specific sequencerequirement, mutations that interfered with thepropagation of W8 were identified by consecu-tive substitution of residues 5–55 of Sup35 byproline and insertion of glycine at alternatesites in this segment. Interestingly, propagatingW8 with single mutations at residues 5–7 andaround residue 43 caused the strain to trans-mute. In contrast to the assertion that [PSI]existed as a dynamic cloud of variants, no ran-dom drift in transmission characteristics wasdetected in mitotically propagated W8

populations. Electron diffraction and mass-per-length measurements indicate that, similar tothe 3 previously characterized strains, W8fibers are composed of about 1 prion moleculeper 4.7-A

�cross-b repeat period. Thus differ-

ently folded single Sup35 molecules, notdimeric and trimeric assemblies, form the basicrepeating units to build the 4 [PSI] strains.

P.112: Using proteinase K to study thestructure of prions

Christopher Silva1,Ester V�azquez-Fern�andez2, and

Jes�us Requena3

1US Department of Agriculture, Albany, CA USA;2University of Alberta, Edmonton, AB Canada;3University of Santiago de Compostela-IDIS;

Santiago de Compostela, Spain

Introduction. The secondary structure ofprions is composed almost entirely of b-sheetsecondary structure. Structural constraints sug-gest that the b-sheet secondary structure isarranged in a b-solenoid. The b-sheet second-ary structure is thought to be responsible forthe remarkable resistance prions have to pro-teinase K (PK) digestion. A detailed analysis ofthe PK digestion products can be used to studythe structure of prions by identifying the loca-tion of PK cleavage sites in wild type and GPI-anchorless prions.

Materials and Methods. Mass spectrometryand antibodies were used to identify PK cleav-age sites in PrPSc. The PK cleavage sites(from 23–160) in the wild type 263K anddrowsy strains of hamster adapted scrapiewere identified. In addition murine-adaptedGPI-anchorless prions were digested with PKand analyzed by mass spectrometry to deter-mine the PK cleavage sites throughout theentire protein. Antibody-based analysis wasperformed by other researchers to determinethe PK cleavage sites in human and hamsterprions.


Results and Conclusions. The results ofthese diverse analyses were used to identify theregions of the prion that were accessible to PKdigestion. These regions were presumed to beaccessible to PK due to flexible stretches con-necting the b-strand components in PrPSc.These data, combined with physical constraintsimposed by spectroscopic results, were used topropose a qualitative model for the structure ofPrPSc. Assuming that PrPSc is a four rungb-solenoid, we have threaded the PrP sequenceto satisfy the PK proteolysis data and otherexperimental constraints.

P.113: The chemistry of prions: Smallmolecules, protein conformers and

mass spectrometry

Christopher Silva andMelissa Erickson-Beltran

US Department of Agriculture; Albany, CA USA

Introduction. Prions propagate by convertinga normal cellular isoform (PrPC) into the prionisoform (PrPSc) in a template-driven process.The lysines in PrPC are highly conserved andstrongly influence prion propagation, based onstudies using natural polymorphisms of PrPC

and transgenic animals expressing natural andunnatural PrPC polymorphisms.

Materials and Methods. Prions from differ-ent hamster-adapted prion strains were reactedwith small molecule reagents. The extent ofthis reaction was quantitated by mass spectrom-etry or Western blot-based analysis. Some sam-ples were analyzed to quantitate the loss ofinfectivity associated with the correspondinglysine acetylation.

Results and Conclusions. The reactivity ofeach of the prion strains with a given reagentwas different. The strains showed differencesin the reactivity of the N-terminal lysines, theC-terminal lysine and some of the lysines in thetwo large a-helicies. Western blotting showeddifferences in the lysine that was part of the

epitope of the mAb 3F4. In addition, a relation-ship between the extent of the reaction oflysines and the loss of infectivity was observed.The approach of using differences in chemicalreactivity can be used to understand the role ofother amino acids in prion replication. In addi-tion this approach can be used to understandthe role that lysines play in the propagation ofother prion-like protein misfolding diseasessuch as AD, ALS, and PD.

P.114: Using patient-specific fibroblastsand iPSC-derived neurons to uncovercellular phenotypes associated with

prion diseases

Jue Yuan1, Leslie Cooperman1,Christina Orru2, Dongyun Han1,

Hisashi Fujioka1, Elizabeth Shick1,Yi-An Zhan1, Mark Rodgers1,Robert Wyza1, Brian Appleby1,

Miguel Quinones-Mateu1, Shulin Zhang1,Tingwei Mu1, Byron Caughey2, Xin Qi1,

Paul Tesar1, and Wen-Quan Zou1

1Case Western Reserve University; Cleveland, OH

USA; 2NIH/NIAID Rocky Mountain Laboratories;

Hamilton, MT USA

Cell biology of prion formation and spreadremains incompletely understood, largelybecause of lack of authentic cell models. Wereport isolation of fibroblasts from skin tissues,derivation of induced pluripotent stem cells(iPSCs), differentiation of iPSCs into matureneurons, and detection of disease-related pheno-types in fibroblasts and iPSC-derived neurons.Fibroblasts were isolated from skin samples of23 subjects including 9 asymptomatic subjectscarrying 6 different PrP mutations, 4 patientswith sporadic CJD (sCJD), and 10 normal con-trols. Surprisingly, not only protease-resistantPrP was detected with Western blotting but alsoseeding activity was detected with RT-QuIC infibroblasts of some PrP mutation carriers orsCJD patients. After iPSCs were derived fromfibroblasts of mutation carriers (E200K orD178N) and normal controls, functional mature


iPSC-derived neurons were further differenti-ated, as evidenced by immunostaining with theneuronal marker Map2 and by patch-clamprecording of GABA-induced current. Whilemigration and glycosylation of PrP from fibro-blasts were different from those of brain PrP,iPSC-derived neurons exhibited the PrP profilesimilar to the brain PrP. Notably, shortened neu-rites and neuritic-beading, characteristics ofneurodegeneration, were more readily observedin iPSC-derived E200K neurons or in iPSC-derived neurons challenged with sCJD brainhomogenates compared to neurons derived fromiPSCs of normal subjects. Our study has gener-ated patient-specific fibroblasts and iPSC-derived neurons that exhibit cellular phenotypesand seem to be authentic cell models for prob-ing human prion diseases. [Supported by theCJD Foundation Award, NIHNS087588,NIHNS062787, and bridge funding from Uni-versity Hospitals Case Medical Center.]

P.115: Prion protein protects againstrenal ischemia/reperfusion injury

Bo Zhang1,2,3, Daniel Cowden4,Fan Zhang4,5, Jue Yuan4, Sandra Siedlak4,Mai Abouelsaad4, Liang Zeng4,6, XuefengZhou2, John O’Toole7,8, Alvin S Das4,

Diane Kofskey4, Miriam Warren4, ZehuaBian2, Yuqi Cui2, Tao Tan2, AdamKresak9, Robert E Wyza9, Robert BPetersen4,10,11, Gong-Xian Wang6,

Qingzhong Kong4,10,12, Xinglong Wang4,John Sedor7,8, Xiongwei Zhu4, Hua Zhu2,

and Wen-Quan Zou4,6,10,12,13

1Institute of Organ Transplantation; Tongji

Hospital; Tongji Medical College; Huazhong

University of Science and Technology; Wuhan,

HuBei, China; 2Department of Surgery; Davis

Heart and Lung Research Institute; The Ohio State

University; Columbus, OH USA; 3Key Laboratory

of Ministry of Health and Key Laboratory of

Ministry of Education; Wuhan, HuBei, China;4Department of Pathology; Case Western Reserve

University/University Hospitals Case Medical

Center; Cleveland, OH USA; 5Department of

Neurosurgery; Shandong University; Jinan, China;

6The First Affiliated Hospital; Nanchang

University; Nanchang, Jiangxi Province, The

People’s Republic of China; 7Kidney Disease

Research Center; Case Western Reserve University/

University Hospitals Case Medical Center;

Cleveland, OH USA; 8Departments of Medicine and

Physiology and Biophysics; Case Western Reserve


Center; Cleveland, OH USA; 9Human Tissue

Procurement Facility (HTPF) and the

Comprehensive Cancer Center Tissue Resources

Core; Case Western Reserve University/University

Hospitals Case Medical Center; Cleveland, OH

USA; 10Department of Neurology; Case Western

Reserve University/University Hospitals Case

Medical Center; Cleveland, OH USA; 11Department

of Neuroscience; Case Western Reserve University/

University Hospitals Case Medical Center;

Cleveland, OH USA; 12National Center for

Regenerative Medicine; Case Western Reserve


Center; Cleveland, OH USA; 13National Prion

Disease Pathology Surveillance Center; Case

Western Reserve University/University Hospitals

Case Medical Center; Cleveland, OH USA

The cellular prion protein (PrPC), a proteinmost noted for its link to prion diseases, hasbeen found to play a protective role in ischemicbrain injury. To investigate the role of PrPC inthe kidney, an organ highly prone to ischemia/reperfusion (IR) injury, we examined wild-type(WT) and PrPC knockout (KO) mice that weresubjected to 30-min of ischemia followed by 1,2, or 3 d of reperfusion. Renal dysfunction andstructural damage was more severe in KO thanin WT mice. While PrP was undetectable in KOkidneys, Western blotting revealed an increasein PrP in IR-injured WT kidneys compared tosham-treated kidneys. Compared to WT, KOmice kidneys exhibited increases in oxidativestress markers heme oxygenase-1, nitrotyrosine,and Ne-(carboxymethyl)lysine, and decreases inmitochondrial complexes I and III. Notably,phosphorylated extracellular signal-regulatedkinase (pERK) 1/2 staining was predominantlyobserved in tubular cells from KO mice follow-ing 2 d of reperfusion, a time at which significantdifferences in renal dysfunction, histologicalchanges, oxidative stress, and mitochondrialcomplexes between WT and KO mice were


observed. Our study provides the first evidencethat PrPC may play a protective role in renal IRinjury, likely through its effects on mitochondriaand ERK signaling pathways. [Supported by theNational Institutes of Health (NIH) NS062787,NS087588, the CJD Foundation, and bridgefunding fromUniversity Hospitals Case MedicalCenter to W.Q.Z. and a Scientist DevelopmentGrant (12SDG12070174) from American HeartAssociation to H.Z.]

P.116: Prion protein promotes kidneyiron uptake via its ferrireductase activity

Ajai Tripathi, Swati Haldar, andNeena Singh

Case Western Reserve University; Cleveland, OH

USA

Brain iron-dyshomeostasis is an importantcause of neurotoxicity in prion disorders, agroup of neurodegenerative conditions associ-ated with the conversion of prion protein (PrPC)from its normal conformation to an aggregated,PrP-scrapie (PrPSc) isoform. Alteration of ironhomeostasis is believed to result from impairedfunction of PrPC in neuronal iron uptake via itsferrireductase activity. However, unequivocalevidence supporting the ferrireductase activityof PrPC is lacking. Kidney provides a relevantmodel for this evaluation because PrPC isexpressed in the kidney, and »370 mg of ironare re-absorbed daily from the glomerular filtrateby kidney proximal tubule cells (PT), requiringferrireductase activity. Here, we report thatPrPC promotes the uptake of transferrin (Tf) andnon-Tf-bound-iron (NTBI) by the kidney invivo, and mainly NTBI by PT cells in vitro.Thus, uptake of 59Fe administered by gastricgavage, intravenously, or intraperitoneally wassignificantly lower in PrP-knock-out (PrP¡/¡)mouse kidney relative to PrPC/C controls.Selective in vivo radiolabeling of plasma NTBIwith 59Fe revealed similar results. Expressionof exogenous PrPC in immortalized PT cellsshowed localization on the plasma membraneand intracellular vesicles, and increased trans-epithelial transport of 59Fe-NTBI and to asmaller extent 59Fe-Tf from the apical to the

basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrP 51–89) lacked thisactivity. Furthermore, excess NTBI and hemincaused aggregation of PrPC to a detergent-insol-uble form, limiting iron uptake. Together, theseobservations suggest that PrPC promotesretrieval of iron from the glomerular filtrate viaits ferrireductase activity, and modulates kidneyiron metabolism.

P.117: The multivesicular body is themajor internal site of prion conversion

Lois Greene, Yang-in Yim, Bum-chan Park,Rajgopal Yadavalli, Xiaohong Zhao, and

Evan Eisenberg

National Institutes of Health; Bethesda, MD USA

Conversion of the properly folded prion pro-tein (PrPc) to its insoluble misfolded amyloidform, PrPsc, is thought to be the crucial eventin the pathogenesis of transmissible spongiformencephalopathy. Although there is a consensusthat this process occurs along the endocyticpathway, evidence as to its exact localizationhas so far remained inconclusive. To determinewhich specific endosomal organelle is themajor internal site of prion conversion, wecombined cell imaging with biochemical tech-niques. Treating two chronically prion-infectedcell lines, SMB and ScN2a, with calpain inhibi-tors is known to decrease PrPsc levels, but themechanism of this PrPsc clearance is incom-pletely understood. We found that, before beingcleared from the cells, PrPsc localized toenlarged multivesicular bodies (MVBs). Inter-estingly, the same decrease in PrPsc levels wasobserved when MVB maturation was blockedby knocking down Rab7, Tsg101 or Hrs, orwhen trafficking from the early endosome wasinhibited by overexpressing Rab5 or Rab22.Conversely, PrPsc levels actually increasedwhen traffic out of the MVB was blocked bydisabling the retromer complex through aknock down of Vps26 or SNX2. Crucially,enhanced degradation or perturbations in intra-cellular cholesterol were eliminated as pro-cesses contributing to PrPsc clearance,


indicating that the reduction in PrPsc levels wasdue to its decreased conversion from PrPc, thusdemonstrating that the major internal site ofprion conversion is the MVB.

P.118: The view from above: Thepotential of aerial surveillance in

quantifying CWD infection rates at theherd level

Christopher Silva andMelissa Erickson-Beltran

US Department of Agriculture; Albany, CA USA

Background. Large mammals such as domes-tic cattle and red deer have been reported to alignthemselves with the magnetic North Pole. Sincechronic wasting disease (CWD) affects thebehavior of infected cervids, it may be possibleto estimate the infection rate, at a herd level, bydetermining the changes in the alignment of theanimals with respect to the magnetic pole. Usingavailable aerial surveillance data from Califor-nia, a CWD-free state, the alignment of a freeranging elk was determined.

Materials and Methods. Tomales Point is a2,600 acre fenced-in preserve inside the PointReyes National seashore. It consists of opengrassland and coastal scrub where native Cali-fornia tule elk (Cervus canadensis nannodes)roam freely. These elk were introduced to thepreserve in 1978 and are part of a program torebuild the tule elk population in California.There are no other large mammals inside thefence. The aerial surveillance data was exam-ined and approximately 146 elk were identifiedand their orientation determined.

Results and Conclusions. Analysis of thealignment data demonstrated that tule elk showno particular alignment in any direction. Furtherresearch will need to be performed to determineif CWD infected animals have a greater ten-dency to align themselves with the magneticpole. As the cost of high-resolution imagesdecreases and aerial surveillance data becomes

more available, it may be possible to use otherparameters, such as fawn count per doe to esti-mate CWD prevalence in an infected herd.

P.119: PrP0\0 mice show behavioralabnormalities that suggest PrPC has arole in maintaining the cytoskeleton

Christopher Silva1, Matthais Schmitz2, andInga Zerr1,2

1US Department of Agriculture; Albany, CA USA;2University Medical Center G€ottingen; Department

of Neurology; G€ottingen, Germany

Background. PrPC is highly conservedamong mammals, but its natural function isunclear. Prnp ablated mice (PrP0/0) appear todevelop normally and are able to reproduce.These observations seem to indicate that thegene is not essential for viability, in spite of itbeing highly conserved.

Materials and Methods. A variety of modelswere used to better understand the physiologi-cal role of PrPC. Wild type (WT) and PrP0/0

mice were subjected to a series of standardbehavioral tests to detect phenotypic differen-ces. Tissues from these mice were studied atthe physiological level. Biochemical differen-ces in mouse derived tissues were examined bymass spectrometry.

Results and Conclusions. PrP0/0, but notWT, mice showed a substantially reduced abil-ity to build nests. Age-dependent behavioraldeficits in memory performance, associativelearning, and basal anxiety were also observedin PrP0/0 mice. WT, but not PrP0/0, miceshowed increases in four neurofilament (NF)proteins levels as they aged. Five other proteinswere found to be differentially abundant inolder (18 month) WT but not in PrP0/0 mice.NF-H phosphorylation was reduced in bothPrP0/0 mouse and cell models. PrPC ablationwas associated with the expression of Fyn andphospho-Fyn, a potential regulator for NFphosphorylation. The number of b-tubulin III-


positive neurons in the hippocampus wasdiminished in PrP0/0 mice relative to WT mice.These data indicate that PrPC plays an impor-tant role in cytoskeletal organization, brainfunction, and age-related neuroprotection. Ourwork represents the first direct biochemical linkbetween these proteins and the observed behav-ioral phenotypes.

P.120: A protein polymerization cascademediates the toxicity of seeded amyloidsof non-pathogenic huntingtin in yeast

Genrikh Serpionov, Alexander Alexandrov,and Michael Ter-Avanesyan

Bach Institute of Biochemistry; Moscow, Russia

A distinct group of human neurodegenera-tive amyloidoses, including Huntington dis-ease, is caused by expansion of polyglutamine(polyQ) stretches in several otherwise unrelatedproteins. Studies in yeast, using the first exon ofthe human huntingtin (Htt)-encoding gene,demonstrate that the deleterious effect of Httalso correlates with the length of its polyQ.While an N-terminal fragment of mutant Httwith a stretch of 103 glutamine residues(Htt103Q) aggregates and causes toxicity, itswild type variant with a sequence of 25 gluta-mines (Htt25Q) is not toxic and does not aggre-gate. Here, we observed that non-toxicpolymers of proteins with long polyQ or polyQinterspersed with other residues (polyQX) canseed polymerization of Htt25Q, which causestoxicity. We further showed that toxicity ofHtt25Q is related to the ability of its polymersto seed polymerization of the essential gluta-mine/asparagine (Q/N)-rich Sup35 proteinwhich results not only in depletion of its solu-ble form but also in sequestration of its essen-tial partner protein, Sup45, through its bindingto Sup35 polymers. Prion amyloids of the Q/N-rich Rnq1 protein can also seed Htt25Q poly-merization which is accompanied with appear-ance of Sup35 polymers and growth inhibition.Importantly, just polymers of Htt25Q, butnot of Rnq1 or polyQ/QX, seed Sup35 poly-merization, suggesting that Htt25Q polymersact as intermediators in seeding Sup35

polymerization. The obtained data provide anovel insight into interactions between amyloi-dogenic proteins and pathophysiological inter-relations between various polyQ disorders.

P.121: Improving therapeutic efficacy of2-aminothiazoles in PrP prion disease

Kurt Giles, David Berry, Carlo Condello,Sumita Bhardwaj, Abby Oehler,Alejandra Gallardo-Godoy,

Stephen DeArmond, Adam Renslo, andStanley Prusiner

University of California San Francisco,

San Francisco, CA USA

The lack of therapeutics that halt or slowneurodegenerative diseases is a growing chal-lenge for societies with aging populations. Werecently demonstrated that compounds with the2-aminothiazole (2-AMT) scaffold couldextend survival in PrP prion-infected mice.One of these compounds, IND24, was effectiveagainst the RML strain of mouse-passagedsheep scrapie, and chronic wasting diseaseprions, but did not extend survival in Tg(HuPrP) mice infected with Creutzfeldt-Jakobdisease prions.

Upon studying the brains of RML-infectedmice treated with IND24, we discovered that anew prion strain emerged. Brain homogenatesprepared from RML-infected, IND24 treatedmice were used to infect CAD5 cells. Theseprions were found to be resistant to IND24 atconcentrations up to 20 mM. In attempts toavoid the development of drug resistance, weinvestigated the effect of varying the dose andduration of IND24 treatment of the RML-infected mice. When treatment was started pro-phylactically, IND24 resulted in a four-foldextension in survival over vehicle-treatedcontrols.

In parallel, medicinal chemistry optimizationthe 2-AMT scaffold, supported by pharmacoki-netic analysis in mice, generated compoundswith increased efficacy in cells that reachedhigh concentrations in the brain. Long-termefficacy studies showed up to a three-fold


extension in survival when dosing was startedafter inoculation. In addition, hits from a sec-ond chemical scaffold, the biaryl amides, werealso optimized and subjected to efficacy stud-ies. Neuropathological investigations suggestthat new strains also emerged from other 2-aminothiazoles. Whether some of these com-pounds might act synergistically remains to bedetermined.

P.122: a-synuclein amyloid interactionwith prion protein: A putative overlap of

2 neurodegenerative diseases

Suzana Aulic, Lara Masperone, Elisa Isopi,Joanna Narkiewicz, andGiuseppe Legname

Scuola Internazionale Superiore di Studi Avanzati;

Trieste, Italy

While the function of the cellular form of theprion protein (PrPC) is still under debate, thereare several reports implying PrPC beinginvolved in binding and modulation of the tox-icity of amyloids involved in neurodegenera-tive disorders (such as Ab oligomers inAlzheimer disease). In this work we investi-gated whether this is also true for a-synuclein(a-syn), a protein involved in neurodegenera-tion in Parkinson disease (PD). In this study,we used the same methodology employed toobtain synthetic mammalian prions, to formrecombinant mouse a-syn amyloids. We char-acterized various preparations of a-syn amy-loids (using atomic force microscopy andbiochemical approaches) and subsequentlyexplored the uptake of these preparations inneuroblastoma N2a cells which express PrPC

(N2aWT) and N2a cells knocked out for PrPC

protein (N2aKO). Our results show that theuptake of a-syn amyloids is lower in N2aKO ifcompared to control cells. Confocal micros-copy and co-localization with sub-compart-mental markers revealed that the a-synamyloids co-internalized with PrPC, accumu-lated and trafficked to lysosomes. Furthermore,serial passages of N2aWT cells treated witha-syn amyloids led to sustained accumulation

of both, a-syn and PrP. Further work wasrequired to validate the importance of this inter-action in disease progression in vivo. Thus, weperformed stereotaxic injections in substantianigra pars compacta of a-syn amyloids in FVBPrPWT and FVB PrPKO mice. Our findingssuggest a role for PrPC in regulating of a-synuptake, thus, evidencing a link between the twoneurodegeneration associated proteins. Thisstudy suggests an overlap between prion dis-ease and PD.

P.123: Substitution of histidine bytyrosine at non-OR copper-binding site

promotes prion conversion

Thanh Hoa Tran1, Phuong Thao Mai1,Juan Mar�ıa-Torres2, andGiuseppe Legname1

1Department of Neuroscience; Laboratory of Prion

Biology; Scuola Internazionale Superiore di Studi

Avanzati (SISSA); Trieste, Italy; 2Centro de

Investigaci�on en Sanidad Animal (CISA-INIA);Valdeolmos, Madrid, Spain

Prion protein is a copper binding protein viahistidine residues in the octapeptide repeats(OR) and in the non-OR regions. These struc-tures are located in the disordered N-terminaltail of the protein while no copper binding hasbeen reported in the C-terminal domain.Although the functional implication of copperbinding to PrP is not completely clear, it isbelieved that copper plays an important role inprion diseases. To determine whether coppercoordination modulates prion formation, wecreated several mutant mouse (Mo) PrP con-structs by replacing histidine residues at theOR and non-OR regions by tyrosine. Theseconstructs were transfected into ScN2a cellsand the efficiency of prion conversion wasmeasured. The results showed that replacinghistidines by tyrosines at non-OR region led toincrease of PrPSc levels, while histidinereplacement in the OR did not alter prion con-version in cells. In addition, cuprizone removalof copper did not alter the level of PrPSc inMoPrP H95Y containing cells whereas it


increased in wild-type (wt) MoPrP. To testthese mutants in vitro, we produced recombi-nant proteins and carried out a fibrilizationassay and compared the lag phases durationamong different constructs. The results clearlyshow that the constructs with tyrosine (H95Y,H110Y, or H95,110Y) required shorter lagphases to aggregate compared to wt MoPrP.Based on these data, we could conclude thatsubstitution of histidine by tyrosine residues atnon-OR region can enhance PrPC-PrPSc con-version process, and that the non-OR copper-binding site may possess a critical role in thisprocess.

P.124: Distinct disease phenotypesproduced by a de novo generated

synthetic prion strain: Conformationalinstability before adaptation

Fabio Moda1, Nhat Tran Tanh Le2,Tommaso Virgilio1, Samanta Mazzetti1,

Suzanna Aulic2, Luisa Palamara1,Ilaria Campagnani1, Olivier Andr�eoletti3,

Fabrizio Tagliavini1, andGiuseppe Legname2

1IRCCS Foundation “Carlo Besta” Neurological

Institute; Milan, Italy; 2Scuola Internazionale

Superiore di Studi Avanzati (SISSA); Trieste, Italy;3UMR INRA-ENVT; Physiopathologie Infectieuse et

Parasitaire des Ruminants; Ecole Nationale

V�et�erinaire de Toulouse; Touluse, France

Prions are infectious proteins that possessmultiple self-propagating structures, whichdefine different strains. The structural informa-tion for strain diversity is contained in the fold-ing of the pathological isoform, PrPSc.Following an in vitro protocol, recombinantmouse PrP (recMoPrP) was converted to ultra-structurally different amyloid fibrils withoutany seeding factor. One of these preparations(recMoPrP#4) efficiently propagated in PMCAusing the brains of mice overexpressing PrPC

(Tga20) as substrate. RecMoPrP#4 was able toinfect either GT1 or N2a cell lines causing theconversion of endogenous PrPC to PK-resistant

forms. We next assessed the ability ofrecMoPrP#4 to propagate in vivo after intrace-rebral inoculation in CD1 mice. The animalsdid not show any evident prion-like pathologyand were culled at the end of their lifespan. Thebrain of these mice was either used for (i) a sec-ond passage transmission or (ii) analyzed byPMCA. The latter revealed the presence of PK-resistant PrP with an uncommon biochemicalprofile when compared to that of known murineprion strains. This amplified isolate was intra-cerebrally injected in CD1 mice, which devel-oped disease after a relative short incubationtime (»160 days). Immunohistochemical andbiochemical analysis revealed the presence ofthree different PK-resistant prion isolates ableto produce a subset of completely differentpathologies. The biochemical profiles of theisolates that accumulated in the CNS of theseanimals were distinct from that of the originalamyloid used as inoculum. These results indi-cate that synthetic prions can assume multipleintermediate conformations before adaptingand converging to stable strains.

P.125: Distinct strains of Ab prionsimplicated in rapidly progressive

Alzheimer disease

Mark Cohen, Chae Kim, Tracy Haldiman,Mohamed ElHag, Curtis Tatsuoka,Shulin Zhang, Jonathan Haines,Alan Lerner, and Jiri Safar*

Case Western Reserve University; Cleveland,

OH USA

Because over 75% of phenotypic variance oflate onset Alzheimer disease (AD) remainsunexplained by currently identified risk genes,we aimed to investigate the prion paradigm ofAD, specifically the role of structure of thebrain amyloid b (Ab) in remarkably variablerates of clinical decline. Using a tandem ofnovel biophysical methods, we inventoried andanalyzed conformational structural characteris-tics of Ab in the cortex of 48 cases of sporadicAD with distinctly different disease durations,and correlated the data with clinical profiles


and genetics. In both hippocampus and poste-rior cingulate cortex we identified an extensivearray of distinct Ab42 particles that differ inconformational structure, size, and display ofN-terminal and C-terminal domains. In con-trast, Ab40 present at low levels did not form amajor particle with discernible size, and bothN-terminal and C- terminal domains werelargely exposed. Rapidly progressive cases areassociated with a low frequency of APOEe4 allele and demonstrate considerablyexpanded conformational heterogeneity ofAb42, with higher levels of distinctly struc-tured Ab42 particles composed of 30–100 monomers, and fewer particles composedof <30 monomers. Our data indicate thatAb42 generates in the AD brain a broad spec-trum of different conformational structures -strains - that may have a potentially differenttoxicity and accumulation rate. The linkbetween rapid clinical decline and levels ofAb42 with distinct structural characteristicsargue for prion-like mechanism encoding vari-able propagation tempo and phenotypic charac-teristics of the disease in distinct structures ofAb42.

P.126: Ultrastructural and biochemicalanalyses of prion protein amyloids andthe role of lysine and proline residues in

amyloid formation

Allison Kraus, Bradley R Groveman,Kelsie J Anson, Lynne Raymond,

David W Dorward, Andrew Hughson, andByron Caughey

Rocky Mountain Labs; NIH; Hamilton, MT USA

The pathogenic form of prion protein, PrPSc,is the infectious agent responsible for transmis-sible spongiform encephalopathies (TSEs).Efforts to understand and treat prion diseaseshave been hindered by the absence of a defini-tive PrPSc structure. We have recently detaileda parallel in register intermolecular b sheet(PIRIBS) model in which PrPSc is organized asa b-sheet arranged in a series of tandem

hairpins. Stacked assembly of these hairpinsgives rise to a prion fibril. Ultrastructural analy-ses on prion amyloids using electron micros-copy revealed fibrils resembling a celery stalk,or half-pipe, with fibril widths consistent withthe PIRIBS architecture. Further considerationof the PIRIBS model helped identify a potentialobstacle for fibril assembly, namely, electro-static repulsion between 4 cationic lysine resi-dues contained within a central lysine cluster(CLC) of residues 101–110. Also containedwith this region are proline residues 102 and105 involved in familial Gerstmann-Straussler-Scheinker syndrome. Systematic evaluation ofthe CLC lysine and proline residues indicatedindividual P102 or P105 substitution allowedPrP amyloid formation with different PK-resis-tant amyloid cores, implicating the proline resi-dues in influencing local structure. Combinedsubstitution of P102 or P105 residues withcharge neutralization of the CLC lysines byasparagine substitution resulted in very rapid,template-independent amyloid formation withPK-resistant amyloid cores more reminiscentof infectious PrPSc. Thus, the CLC proline andlysine residues influence the amyloidogenicityof PrP, implicating the CLC as a multifacetedmodulator of PrP conversion. Together, thesestudies provide new ways of conceptualizingthe self-propagating, pathogenic structure ofinfectious TSE prion amyloids.

P.127: Caregiver burden in sporadicCreutzfeldt-Jakob Disease

Brian Appleby1, Mary Edmondson2,Chiadi Onyike3, and Alice Uflacker2

1Case Western Reserve University School of

Medicine; Cleveland, OH USA; 2Duke University

Medical Center; Durham, NC USA; 3Johns Hopkins

University School of Medicine; Baltimore, MD USA

Introduction. Caregiver burden is a well-rec-ognized component of dementia care and iswidely studied in other neurodegenerative con-ditions. To our knowledge, no data existregarding caregiver burden in prion disease.


Method. We conducted a retrospective studyexamining caregiver burden in subjects of theJohns Hopkins Young-Onset and Frontotempo-ral Dementias Clinic (2004–2010). We usedthe Zarit Burden Inventory (ZBI) as the out-come variable for measuring overall caregiverburden and collected data on likely contribu-tory variables including cognitive impairment,functional impairment, neurologicalimpairment, and neuropsychiatric symptoms.

Results. We were able to collate data fromsubjects diagnosed with Alzheimer disease(AD, n D 21), behavioral variant frontotempo-ral dementia (bvFTD, nD 33), language variantfrontotemporal dementia (lvFTD, n D 15), andsporadic Creutzfeldt-Jakob disease (sCJD, n D7). Caregivers for subjects with bvFTD andsCJD exhibited the highest ZBI scores (p D0.026), Neuropsychiatric Inventory-Q (NPI-Q)total severity scores (p D 0.004), and NPI-Qcaregiver distress scores (p D 0.002). In thefinal regression model, differences in ZBIscores among diagnostic groups were notaffected by cognitive, functional, or neurologi-cal impairments. There was an interactioneffect between diagnostic category and NPI-Qtotal severity scores in regard to differences inZBI score, suggesting that much of the differ-ences in ZBI scores were mediated by neuro-psychiatric symptoms.

Conclusion. Caregiver burden is exception-ally high in sCJD, approaching levels observedin bvFTD. Much of differences observed inZBI scores are likely mediated by neuropsychi-atric symptoms. Future studies are needed tostudy caregiver burden in prion disease and itshould be considered a secondary outcomemeasure in future treatment studies.

P.128: Bioassay using ovine and cervidPrP transgenic mice for discriminationof scrapie and CWD origins in sheep

and goats

Sally Madsen-Bouterse1,*,Dongyue Zhuang2, David Schneider2,

Rohana Dassanayake1, Aru Balachandran3,Gordon Mitchell3, andKatherine O’Rourke1

1Department of Veterinary Microbiology and

Pathology; College of Veterinary Medicine;

Washington State University; Pullman, WA USA;2Animal Disease Research Unit; Agricultural

Research Service; US. Department of Agriculture;

Pullman, WA USA; 3National and OIE ReferenceLaboratory for Scrapie and CWD; Canadian Food

Inspection Agency; Ottawa Laboratory–

Fallowfield; Ottawa, ON Canada

As the United States works toward the eradi-cation of scrapie, identifying TSE reservoirs thatcould lead to disease re-emergence is imperative.Development of transgenic mice expressingeither the ovine or cervid prion protein has aidedcharacterization of scrapie and CWD, respec-tively. We hypothesize that transgenic mousemodels will discern whether new incidents ofscrapie in sheep and goats with clinical diseaseoriginated from CWD exposure. Two transgenicmouse lines (Tg338 and TgElk; minimum5 mice/strain) were inoculated with brainhomogenate from clinically affected animalsincluding sheep or goats with naturally acquiredclassical scrapie, white-tailed deer with naturallyacquired CWD (WTD-CWD), or sheep experi-mentally inoculated with elk-CWD (sheepelk-CWD). Transmission was assessed via survivalanalysis and western blot characterization ofbrain PrPres. WTD-CWD transmitted efficientlyto TgElk with all mice culled due to clinical dis-ease, whereas all Tg338 remained asymptomaticat endpoint with no PrPres detected in the brain.Ovine and caprine scrapie transmitted poorly toTgElk with all mice asymptomatic at endpointand 6.8% brain-positive for PrPres, whereas allTg338 were culled due to clinical disease. Shee-pelk-CWD yielded Tg338 that were all


asymptomatic at endpoint and were all brain-positive for PrPres. However, sheepelk-CWD

yielded TgElk with 5/22 displaying clinical dis-ease near endpoint but 16/22 brain-positive forPrPres. Furthermore, TgElk-PrPres molecularmass appeared lower when inoculated with cap-rine scrapie versus WTD-CWD and both molec-ular masses were yielded when inoculated withsheepelk-CWD. These findings suggest primarypassage in Tg338 and TgElk could discernwhether scrapie in sheep and goats originatedfrom CWD exposure.

P.129: Elucidating the role of matrixmetalloproteases in prion

protein proteolysis

Victoria Lewis1, Vanessa Johanssen1,Amber Lothian2, Blaine Roberts2,Peter Crouch1, and Steven Collins1,3

1Department of Pathology, The University of

Melbourne, Parkville, Victoria, Australia; 2The

Florey Institute of Neuroscience and Mental Health,

Parkville, Victoria, Australia; 3Department of

Medicine, RMH, The University of Melbourne,

Parkville, Victoria, Australia

Like many proteins, the cellular prion pro-tein, PrPC, undergoes regulated and constitu-tive endoproteolysis. These post-translationalevents include well described alpha- and beta-cleavage, newly described gamma-cleavage,and protease and phospholipase mediated PrPCshedding. The exact purpose of PrPC endopro-teolysis is not determined, with increasing evi-dence for different biological roles of thevarious N- and C-terminal fragments produced,as well as the full-length protein.

The major PrPC proteolytic cleavage event isalpha-cleavage, and often there is higher expres-sion of the C-terminal cleavage fragment, C1,than full-length PrPC. The exact identity of theprotease responsible for alpha-cleavage is con-tentious, with strong evidence both for andagainst the involvement of the ADAMs (a disin-tegrin and metalloprotease) family of proteases.Importantly, this alpha-cleavage event has beenlinked to prion diseases.

Using various in vitro assays, we have identi-fied the matrix metalloprotease (MMP) familyof proteases as key regulators of PrPC endopro-teolysis, particularly at the alpha-cleavage site.We have determined MMP2 to be an “alpha-PrPase”, producing C1 from full-length humanPrP in a recombinant protein assay. Surpris-ingly, treating cultured cells with a pan-MMPinhibitor (prinomastat, selective for MMPs-2, 3,9, 13 and 14) increased cell associated C1 lev-els, highlighting the complex and dichotomousnature of the MMPs in the regulation of alpha-cleavage. However, consistent with this C1increase, we observed significantly decreasedPrPSc levels in prinomastat treated prioninfected cells. Our findings provide new insightinto the normal biology of PrPC proteolyticprocessing, as well as a potential new avenuefor therapeutic interventions in prion disease.

P.130: PK-induced disassembly ofpartially unfolded PrPSc fibers supports

a multi-rung architecture ofPrPSc subunits

Alejandro M Sevillano1, Matthijn Vos2,Ilia Baskakov3, and Jesu�s R Requena1,*

1CIMUS Biomedical Research Institute &

Department of Medicine; University of Santiago de

Compostela-IDIS; Santiago de Compostela, Spain;2The FEI Company; Eindhoven, The Netherlands;

3Center for Biomedical Engineering and

Technology; Department of Anatomy and

Neurobiology; University of Maryland School of

Medicine; Baltimore, MD USA

Evidence based on solid state NMR stronglysuggests that recombinant PrP (rPrP) amyloidfibers are an in-register stack of single-rung“flat” monomers, whose b-strand rich coresspan from position »160 to the C-terminus. Incontrast, evidence from, 2D electron crystalog-raphy, X-ray fiber diffraction, and cryo-elec-tron microscopy and helical reconstructionsuggests that PrPSc consists of stacks of 4-rungb-solenoids. However, the recently proposedPIRIBS model argues that PrPSc monomers are


also flat, with rPrP amyloid-like b-strand richcores extending up to position »90.

Besides a proteinase K (PK) resistant corespanning »90–230, PrPSc has an inner “super-resistant” »152–230 core that resists partial,reversible unfolding induced by guanidine. Wetherefore reasoned that if PrPSc is a multi-rungsolenoid, PK-treatment of partially unfoldedPrPSc fibers should necessarily result in theircomplete disassembly, as the N-terminal “base”of each monomer desintegrates. In contrast, ifPrPSc monomers are flat, fibers would persistafter such treatment, as seen when rPrP fibersare treated with PK under conditions that pre-serve their C-terminal b-strand rich cores,which remain stacked.

Our results show virtually complete disas-sembly of partially unfolded PrPSc fibers afterPK treatment, supporting a multi-rung, ratherthan flat, architecture of PrPSc monomers. As acorollary, PrPSc fibers would be made up of 2protofilaments, as has been the general consen-sus based on the general appearance of negativestain TEM images. In this respect, we presentevidence suggesting that fiber segments with a“half-pipe” morphology present in PrPSc prepa-rations, suggestive of wide, single-protofila-ment fibers, might be tubulin impurities.

P.131: Sinergistic effects of 2 anti-prioncompounds targeting the cellular prion

protein

Tania Massignan1,5, Claudia Stincardini1,5,Ilaria Vanni2, Nunzio Iraci3,

Matteo Stravalaci1, Alessandro Negro4,Claudia D’Agostino2, Marco Gobbi1,Laura Colombo1, Romolo Nonno2, and

Emiliano Biasini1,5

1Mario Negri Institute for Pharmacological

Research, Milan, Italy; 2Istituto Superiore di Sanit�a,Rome, RM, Italy; 3University of Perugia, Perugia,

Italy; 4University of Padova, Padova, Italy; 5Center

for Integrative Biology; University of Trento,

Trento, Italy

Mounting evidence suggests that PrPSc couldbe a difficult pharmacological target in prion

diseases. The discovery of therapeutics capableof reducing PrPSc aggregates and halt neurode-generation has so far been unsuccessful. Thestructure of PrPSc is poorly defined, and likelyto be conformationally heterogeneous, as indi-cated by the existence of prion strains. The lat-ter represents a relevant problem for drugdiscovery, as several anti-prion compoundsidentified so far are strain-specific. Moreover,unknown, misfolded PrP intermediates couldbe responsible for the neurodegenerative pro-cess occurring in prion diseases.

We are seeking to develop a pharmacologi-cal strategy that may overcome these problemsby targeting PrPC instead of PrPSc. Since theytarget the native substrate of prion replicationreactions, PrPC-directed compounds could bestrain-independent, and also prevent theappearance of any neurotoxic PrP species.

Here, we started to explore this strategy bycharacterizing 2 known PrPC-directed com-pounds, a cationic tetrapyrrole [Fe(III)-TMPyP] and chlorpromazine hydrocloride(CPH). By employing a variety of biochemicaland biophysical techniques, we confirmed thatFe(III)-TMPyP binds to PrPC with sub-micro-molar affinity. Conversely, we found that CPHfails to bind PrPC at biologically relevant con-centrations. Instead, this compound exerts anti-prion effects by inducing the internalization ofPrPC from the cell surface. Consistent withtheir different mode of action, we found that Fe(III)-TMPyP and CPH act synergistically toinhibit the cytotoxic effects of a PrP mutant,and to block prion propagation in cell cultures.

Our data suggest that targeting PrPC withmultiple, mechanistically distinct approachescould be an effective strategy against priondiseases.


P.132: Hematological shift but noevidence of immunological impairmentin goat kids naturally devoid of the

cellular prion protein (PrPC)

Malin R Reiten*, Maren K Bakkebø,Michael A Tranulis, Arild Espenes, and

Preben Boysen

Norwegian University of Life Sciences; Campus

Adamstuen; Oslo, Norway

Despite extensive research, the physiologicalrole of the cellular prion protein (PrPC) is stillunclear. A nonsense mutation at codon 32 inthe PRNP gene of the Norwegian Dairy Goatbreed results in an early termination of PrPC

synthesis and renders animals homozygous forthe mutation devoid of PrPC. Hence, these PrPC

free animals, unaffected by genetic confound-ers, provide a unique model for studying PrPC

physiology and function. Here, we reportresults of hematological and immunologicalanalyses of goat kids without PrPC (PRNPTer/

Ter), heterozygotes (PRNPC/Ter) and normal(PRNPC/C) PrPC expression. We found thatPRNPTer/Ter kids had an elevated number of redblood cells (RBCs), although within referencevalues. Additionally, RBC volumes wereslightly decreased, while neutrophil numberswere increased, strikingly similar to what wasdescribed in 10 months old transgenic PrPC

KO cattle. Morphological investigations ofblood smears and bone marrow imprintsappeared normal. Studies of fundamentalimmunological parameters such as the relativecomposition of peripheral blood mononuclearcells (PBMCs) and functional studies of macro-phages (phagocytic ability) and T lymphocytes(proliferation after stimulation) demonstratedno significant differences between the PRNPgenotypes. The cell surface PrPC levels onPBMCs correlated with the PRNP mRNAexpression levels and were halved in PRNPC/

Ter and absent in PRNPTer/Ter cells, suggestingthat in PRNPTer/Ter cells, nonsense mediatedmRNA decay occurs. In summary, our resultsindicate a role for PrPC within the bone

marrow, more precisely related to maturationand release of erythroid and granulocytic cells.

P.133: Prion-like characteristics of thebacterial protein Microcin E492

Mohammad Shahnawaz, Kyung-won Park,Rodrigo Diaz-Espinoza, and Claudio Soto

Mitchell Center for Alzheimer Disease and Related

Brain Disorders; Department of Neurology;

University of Texas Medical School; Houston,

TX USA

Microcin E492 (Mcc) is a low molecularweight pore-forming bacteriotoxin. Active Mccis produced only during the exponential phaseof growth, whereas Mcc activity is inhibited atthe stationary phase by formation of amyloid-like aggregates in the culture similar to thoseassociated with many human diseases. Weshow, in a similar manner as prions, Mcc natu-rally exists as two 2 conformers: a b-sheet-rich,protease-resistant, aggregated, inactive form(Mccia), and a soluble, protease-sensitive,active form (Mcca). Exogenous addition of cul-ture medium containing Mccia or purified invitro-generated Mccia into the culture of bacte-ria producing Mcca induces the rapid and effi-cient conversion of Mcca into Mccia, which ismaintained indefinitely, changing the bacterialphenotype. Mccia prion-like activity is confor-mation-dependent and could be reduced byimmunodepleting Mccia. Furthermore, using ayeast reporter assay, Mcc was able to confer aheritable change in phenotype when fused tothe MC region of Sup35p. Interestingly, aninternal region of Mcc shares striking sequencesimilarity with the central domain of the prionprotein that has been shown to be a key to theformation of mammalian prions. A syntheticpeptide spanning this sequence forms amyloid-like fibrils in vitro and is capable of inducingthe conversion of Mcca into Mccia in vivo, sug-gesting that this region correspond to the priondomain of Mcc. Our findings indicate that Mccis the first prokaryotic protein with prion prop-erties that harnesses prion-like transmission toregulate protein function, suggesting that


propagation of biological information usingprion-based conformational switch is an evolu-tionary conserved mechanism.

P.134: Discussing worries and concernsabout prion disease with family members

of patients and unaffected anxiouspeople: From clinical counseling

experience in Japan

Chieko Tamura

Fetal Medicine Clinic; Tokyo, Japan

From nine years of experience through theactivities of the prion disease research group inJapan, we have learned what kinds of prion-dis-ease-related psychosocial issues people have.Although their psychological pain isimmensely large due to the unexpected difficultcondition, most of the people have ability topsychologically adjust to the circumstancesover time, and usually, ordinary psychologicalcounseling theories and techniques are useful.However, we have experienced some psycho-logically “difficult” cases. Some family mem-bers have communication problems withmedical professionals, which may be solvableby carefully answering their questions and pro-viding good information. People’s psychologi-cal distress may take the form of anger towardhospital staff. People who are not motivated topursue the psychological adjustment processare also difficult, but, education about psycho-logical process may be able to motivate them.Also, when prion disease evokes some familialproblems and tension, it may be not easy to besolved. One of the most difficult example is ahypochondriac case. We have experiencedthose people, who are not affected with priondisease, but are unreasonably worried becausethey think there is a possibility that they gotinfected with prion from food, biological drugs,etc. Information provision and ordinary psy-chological counseling skills are not very help-ful for these people. In this presentation, wewould like to review the psychological issuesof people who face the prion-disease-relatedsituation, and discuss how we can provide

psychosocial support and help, including theway how we deal with “difficult” cases.

P.135: Molecular dynamics simulationstudies of novel Q212H, V203G, andN173K mutations in prion diseases

Sara Amidian2, Mohammad Riazi1,Mohd Shahir Shamsir1, and Holger Wille2

1University of Alberta; Edmonton, Canada;2Universiti Teknologi Malaysia; Johor

Bahru, Malaysia

Prion diseases in humans are grouped basedon whether they are sporadic, inherited, oracquired. In inherited prion disease, abnormalprion proteins (PrP) are produced through agenetic mutation of which 40 point mutationshave been discovered. Three novel mutationsV203G, Q212H and N173K have beenreported, but remained questionable whetherthe mutations caused the prion disease. In thisresearch, we preformed molecular dynamicssimulations and structural analysis to investi-gate if these novel CJD related mutationsbehave similarly to known disease causingmutations in the same region of the protein.The results show similar dynamic behavior topathogenic mutations V203I and Q212P, butdiffer when compared to the non-pathogenicN171S polymorphism. All three mutationsV203G, Q212H and N173K showed a decreasein the protein’s overall stability, an slightincrease in flexibility, a major loss in saltbridges in the first and second helix, changes inthe electrostatic surface of PrP and an increasein the solvent exposure of the protein, all ofwhich are common dynamic behaviors ofamong pathogenic prion mutations.


P.136: Mother to offspring transmissionof CWD—Detection in fawn tissues using

the QuIC assay

Amy Nalls, Erin McNulty, Clare Hoover,Jeanette Hayes-Klug, Kelly Anderson,

Edward Hoover, and Candace Mathiason


To investigate the role mother to offspringtransmission plays in chronic wasting disease(CWD), we have employed a small, polyestrousbreeding, indoor maintainable cervid model,the Reeves’ muntjac deer. Muntjac doe wereinoculated with CWD and tested positive bylymphoid biopsy at 4 months post inoculation.From these CWD-infected doe, we obtained 3viable fawns. These fawns tested IHC-positivefor CWD by lymphoid biopsy as early as 40 dpost birth, and all have been euthanized due toclinical disease at 31, 34 and 59 months postbirth. The QuIC assay demonstrates sensitivityand specificity in the detection of conversioncompetent prions in peripheral IHC-positivetissues including tonsil, mandibular, partotid,retropharyngeal, and prescapular lymph nodes,adrenal gland, spleen and liver. In summary,using the muntjac deer model, we have demon-strated CWD clinical disease in offspring bornto CWD-infected doe and found that the QuICassay is an effective tool in the detection ofprions in peripheral tissues. Our findings dem-onstrate that transmission of prions frommother to offspring can occur, and may beunderestimated for all prion diseases.

P.137: Non-adaptive prion amplification(NAPA): Interspecies prion propagationliberated from the constraints of species-

specific PrP primary structure

Jifeng Bian1, Vadim Khaychuk1,Jason Bartz2, Joaquin Castila3,Sehun Kim1, and Glenn Telling1

1Colorado State University; Fort Collins, CO USA;2Creighton University; Omaha, NE USA; 3Parque

Tecnol�ogico de Bizkaia; Derio, Bizkaia, Spain

Conventional wisdom holds that optimalprion disease progression requires related PrPSc

and PrPC primary (10) structures. Accordingly,barriers to transmission of prions from a partic-ular species are generally overcome by expres-sion of PrP from that species in mice. Incontrast, interspecies transmissions are com-monly characterized by low attack rates/long,variable incubation times on 10 transmission,followed by relatively facile transmission onserial passage. Here we explore a distinct vari-ety of host range modifications as prions transitbetween mice expressing different PrP primarystructures which challenge these long held,widely accepted notions. We refer this type ofinterspecies transmission to as non-adaptiveprion amplification (NAPA). In contrast toadaptive prion replication, during NAPA,prions composed of recipient PrPSc formedafter overcoming a species barrier not only failto further propagate in the recipient species, butremarkably retain the host range properties ofprions from the original species, despite signifi-cant 10 structural differences. Since our find-ings apply not only to experimentally-adaptedprion strains, but also to naturally occurringprions, including transmissible mink encepha-lopathy and scrapie, they signify greater hostrange malleability and more frequent asymp-tomatic prion replication during interspeciestransmissions than previously thought. Of par-ticular significance, previous studies describingvCJD/BSE transmissions to mice expressinghuman or bovine PrP that were hard to under-stand in the context of adaptive prion replica-tion, are reconciled within the framework ofNAPA. Further exploration of this phenomenon


is therefore essential in order to refine futureassessments of zoonotic risk, and to fullyunderstand prion transmission mechanisms.

P.138: Coexistence of classical andCH1641-like scrapie prions in

experimentally scrapie-affected sheep

Kohtaro Miyazawa, Kentaro Masujin,Hiroyuki Okada, Yuichi Matsuura, and

Takashi Yokoyama

Influenza and Prion Disease Research Center;

National Institute of Animal Health; NARO;

Tsukuba, Ibaraki, Japan

Scrapie is a prion disease in sheep and goats.Abnormal prion protein (PrPSc) of a few natu-rally-occurring scrapie cases in sheep is remi-niscent of CH1641 scrapie isolate, which ischaracterized by a lower molecular mass ofunglycosylated form of PrPSc as compared tothat of classical scrapie. We have also reportedthat CH1641-like scrapie prion appears afterintraspecies transmission of classical scrapieprion. However, it is still unknown whetherstrains occur a real mutation process or somestrains already existed in the inoculum. In thisstudy, we ask if classical and CH1641-likescrapie prions coexist in sheep. Brains andspleens taken from sheep experimentallyaffected with classical or CH1641-like scrapiewere homogenized and intracerebrally inocu-lated into wild-type and transgenic mice(TgOvPrP59) overexpressing the ovine prionprotein (A136R154Q171 sequence). By westernblot analysis, CH1641-like specific PrPSc pro-files were confirmed in TgOvPrP59 mice inocu-lated with sheep brain homogenate affectedwith CH1641-like scrapie. Wild-type miceinoculated with the same brain homogenatenever developed prion disease. Surprisingly,wild-type mice inoculated with sheep spleenhomogenate affected with CH1641-like scrapiedeveloped the disease. TgOvPrP59 mice inocu-lated with the same spleen homogenate devel-oped 2 different forms of scrapie. DiseasedTgOvPrP59 mice with a relatively shorter incu-bation period accumulated CH1641-like

specific PrPSc. While the mice with a longerincubation period accumulated classical scrapiespecific PrPSc. Taken together, these resultssuggest the coexistence of classical andCH1641-like scrapie prions in spleen of sheepexperimentally affected with scrapie.

P.139: Tissue distribution and in uterotransmission of chronic-wasting disease-associated prions in free-ranging Rocky

Mountain elk

Anca Selariu1, Jenny G Powers2,Margaret A Wild2, Monica Brandhuber1,Amber Mayfield1, Stephenie Fullaway1,Amy Nalls1, Edward A Hoover1, and

Candace K Mathiason1

1Prion Research Center; Department of

Microbiology, Immunology, and Pathology; College

of Veterinary Medicine and Biomedical Sciences;

Colorado State University; Fort Collins, CO USA;2National Park Service; Biological Resources

Division; Fort Collins, CO USA

The presence of disease-associated prions intissues and bodily fluids of chronic wastingdisease (CWD)-infected cervid has receivedmuch investigation, yet little is known aboutCWD mother to offspring transmission. Ourprevious work demonstrated that mother tooffspring transmission is efficient in anexperimental setting (34). To address thequestion of relevance in a naturally-exposedfree-range population, we have assessedmaternal and fetal tissues derived from 19elk dam-calf pairs harvested from RockyMountain National Park (RMNP), a knownCWD endemic region. Conventionalimmunohistochemistry (IHC) identified 3/19CWD positive dams, whereas a more sensitiveassay – the serial protein misfolding cyclicamplification (sPMCA) – detected cervidprions (PrPCWD) in 15/19 dams. PrPCWD tissuedistribution, as demonstrated by sPMCA, waswidespread and included the central nervoussystem (CNS), lymphoreticular system (LRS),reproductive, secretory, excretory and adiposetissues. Interestingly, 5 of the 15 sPMCA


positive dams showed no evidence of PrPCWD

in either CNS or LRS, sites typically assessedin diagnosing CWD. Analysis of fetal tissuesharvested from the 15 sPMCA positive damsrevealed PrPCWD in 80% of fetuses (12/15),regardless of gestational stage. These findingsdemonstrate that PrPCWD is more abundant infree-range elk peripheral tissues than currentdiagnostic methods suggest, and thattransmission of prions from mother to offspringmay contribute to the efficient transmission ofthe CWD in native cervid populations.

P.140: Profiling infectious elk prionprotein distribution based on size using

asymmetrical flow field-flowfractionation

Jitendra Kumar1,2, Stephanie Czub3,Sabine Gilch4, and Valerie Sim1,5


University of Alberta, Edmonton, AB, Canada;2Department of Medicine; Faculty of Medicine &

Dentistry; University of Alberta, Edmonton, AB,

Canada; 3Canadian Food Inspection Agency

Lethbridge Laboratories, Lethbridge, AB, Canada;4Department of Ecosystem and Public Health;

Faculty of Veterinary Medicine; University of

Calgary, Calgary, AB, Canada; 5Neurosciences and

Mental Health Institute; University of Alberta,

Edmonton, AB, Canada

The separation of macromolecules in prion-infected biological samples is a challengingtask. Size exclusion chromatography (SEC) isnot suitable due to irreversible adsorption onthe solid supports of the column packing mate-rials. Asymmetrical flow field-flow fraction-ation (AF-FFF) is a method of choice, whichyields size fractionation similar to SEC andavoids adsorption during separation. The tech-nique allows the separation of particles rangingin size from a few nanometers to severalmicrons. The sizes of prion protein oligomersare considered to drive the toxicity within thebrain of infect animals.

We injected elk brain infected with chronicwasting disease (CWD) through an AF-FFFconnected to a UV-Vis variable wavelengthspectrophotometeter, refractive index detector,multi-angle light scattering (MALS) anddynamic light scattering detector connected tothe 140� angle of the MALS detector. Alter-ation of cross-flow and detector flow parame-ters enabled selective isolation of differentparticle sizes. Fractions were collected and ana-lyzed for prion protein using dot blot. Corre-sponding particle size was calculated fromcumulant analysis of scattered light intensities.Despite the broad size, shape, and compositionof particles present in the whole brain homoge-nate, particles can be selectively fractionatedby AF-FFF. Adjusting cross-flow parameters isan easy way to adjust separation based on spe-cific characteristics of a brain preparation orparticle of interest. Our results suggest that thetechnique is applicable to profile the oligomericdistribution with in brain preparation and couldbe extended to find out the differences betweenstrains and species affected by prionaggregation.

P.141: The role of neuroprotectivechaperones in proteinmisfolding diseases

Julien Donnelier and Janice Braun

University of Calgary; Calgary, Alberta, Canada

The misfolding, aggregation and accumula-tion of proteins is known to be the causal eventin many neurodegenerative disorders. Undernormal conditions a network of chaperones andproteolytic systems fight against the toxiceffects of aberrant proteins. Multiple lines ofevidence indicate that an imbalance betweenthe production and clearance of proteins ini-tiates protein misfolding, synaptic dysfunction,synaptic loss and neurodegeneration. To beginto address why molecular chaperones do notprotect against the cascade of pathogenicevents in neurodegeneration, we have evaluatedthe chaperone network in 5XFAD mice, asevere model of Alzheimer disease as well as


mice lacking the chaperone cysteine string pro-tein (CSPa) a model of fulminant neurodegen-eration. 5XFAD mice overexpress mutanthuman amyloid precursor protein (APP(695))with the Swedish (K670N, M671L), Florida(I716V), and London (V717I) mutations aswell as human presenilin1 (PS1) harbouring,M146L and L286V that cause familial Alz-heimer disease rapidly accumulate Ab42 andhave impaired memory (Oakley et al., 2006).CSPa deficient mice are normal at birth, butpostnatally develop an impairment of synapticfunction in an activity-dependent manner fol-lowed by a fulminant form of neurodegenera-tion, paralysis and early death betweenpostnatal days 40–60 (Fernandez-Chacon et al.,2004). We have found that an overall collapseof the molecular chaperone network does notoccur until very late in the sequence of neuro-degenerative events while compensatory mech-anisms involving upregulation of specificneuroprotective proteins are at play in someneurodegenerative disorders.

P.142: Chronic wasting disease (CWD)transmission into hamsters

Elizabeth Triscott,Camilo Duque-Velasquez, Judd M Aiken,

and Debbie McKenzie

Center for Prions and Protein Folding Diseases;


KEYWORDS. chronic wasting disease,interspecies transmission, prion strains

Chronic wasting disease (CWD) is a conta-gious prion disease of cervids. The continuedexpansion of the disease in North America isresulting in the increasing number of mamma-lian species exposed to this infectious agent. AsCWD is able to infect multiple cervid species,it is likely that variation of the agent mightoccur, due to PrP polymorphisms within andbetween cervid species. Using Syrian Goldenhamsters as a model for interspecies transmis-sion, we infected the hamsters with geneticallydefined CWD isolates from white-tailed deer as

well as with hunter-harvested mule deer andwhite-tailed deer from Saskatchewan. Themajority of the CWD isolates resulted in suc-cessful transmission to hamsters. Biochemicaland neuropathological analyses suggests differ-ences between the CWD isolates.

P.143: In-silico rational design andoptimization of small drug leads for

inhibition of prion-like propagation ofSOD1 misfolding

Vijaya Kumar Hinge1,2, Nikolay Blinov1,2,Neil Cashman3, and Andriy Kovalenko1,2

1Department of Mechanical Engineering;

University of Alberta; Edmonton, Canada;2National Institute for Nanotechnology; National

Research Council of Canada; Edmonton, Canada;3Department of Medicine; University of British

Columbia; Vancouver, Canada

Amyotrophic lateral sclerosis (ALS) is afatal neurodegenerative disease characterizedby the formation of abnormal aggregates ofsuperoxide dismutase 1 (SOD1) enzyme in neu-ronal cells. Misfolding and aggregation ofSOD1 due to mutations, environmental stress,demetalation or overexpression may causeSOD1 to gain toxic properties, which conse-quently leads to death of motor neurons anddeath of ALS patients within 2 to 5 y. Cur-rently, there is no cure for ALS and drugs canincrease the longevity of ALS patients only bya small fraction. The ultimate goal of our studyis to predict molecular structures of misfoldedSOD1 and SOD1 aggregates based on struc-tural restrains derived from the experimentaldata on conformational antibody recognition ofneurotoxic SOD1 species [Neil Cashman lab,University of British Columbia]. These struc-tures will be used as targets for therapeuticintervention in ALS. A new platform for therational drug design enforced by the moleculartheory of solvation (aka 3D-RISM-KH), alongwith the proprietary protocols for optimizationof the delivery of drugs to the brain will beused to support development of new conforma-tional antibodies and drugs against ALS. Along


with the potential for selecting new drugs forclinical studies, this will help to better under-stand the mechanisms of misfolding and oligo-merization of SOD1. In addition, we areinterested in understanding the prion-likemechanisms involved in ALS progressionwhich can provide the best targets for theblockage of the disease’s progression.

P.144: Transmission of CWD to non-human primates: Interim results of a 6

year risk assessment study on thetransmissibility to humans

Bianka Mussil1, Dirk Motzkus1,Georgia Hesse1, Sabine Borchert1,

Barbara Schiller1, Christiane Stahl-Hennig1

, Walter Schulz-Schaeffer2,Michael Beekes3, Martin Daus3,

Hermann M Schatzl5, Sandor Dudas4,Jianmin Yang4, Jean-Philippe Deslys6, and

Stefanie Czub4,5

1German Primate Center; Goettingen, Germany;2Faculty of Medicine; Department of

Neuropathology; Goettingen, Germany; 3Robert

Koch Institute; Berlin, Germany; 4University of

Calgary; Faculty of Veterinary Medicine; Calgary,

Canada; 5Canadian and OIE ReferenceLaboratories for BSE; Canadian Food Inspection

Agency Lethbridge Laboratory; Lethbridge,

Alberta, Canada; 6Commissariat �a l’EnergieAtomique; Fontenay-aux-Roses, France

Rapid spread and high prevalence of CWDin North American captive and free-rangingcervids have raised concerns about a potentialrisk to human health. Evidence exists that skel-etal muscles might harbor significant amount ofprion infectivity which is of great importanceto consumers of venison, velvet and other cer-vid products. In order to assess the risk of pri-mary CWD-transmission, cynomolgusmacaques (Macaca fascicularis) were inocu-lated with high-titer brain homogenates ofCWD-infected white-tailed deer material byintracerebral, intragastric and dermal scarifica-tion routes. Another group obtained a total

amount of 5 kg CWD-positive muscle homoge-nate using a repeated low-dose regimen (eachreceived »125 applications of 40 g musclehomogenate over a 3 y period). Risk of second-ary CWD-transmission via blood or blood-derived products is judged by blood transfusionof monkey-adapted CWD to naive recipients.Based on the in vitro conversion of recombi-nant prion protein, a real-time quaking-inducedconversion (RT-QuiC) assay was optimized byusing lymph node tissues, cerebrospinal fluidsamples and brain homogenate derived fromBSE-inoculated macaques. Results have shownrobust, sensitive, specific detection, high inter-and moderate intra-assay variances in samplesderived from BSE-infected macaques. So far,all analyzed samples from CWD-inoculatedmacaques did not reveal any seeding activity.Future findings of our risk assessment studywill greatly contribute to policy decisionsincluding monitoring of human blood products,CWD surveillance and CWD control in captiveand free-ranging cervids. Here we will presentan update on the current state of the ongoingproject.

P.145: Using yeast as a model tounderstand the mechanisms that

underlie protein aggregation, amyloidformation, and prionization

Stephanie Valtierra, Zhiqiang Du, andLiming Li

Northwestern University; Chicago, Il USA

Current knowledge of prion biology hasbeen greatly enhanced by studies in Saccharo-myces cerevisiae, which contains several epige-netic elements known as yeast prions. The yeastprion [SWIC], whose protein determinant isSwi1, a subunit of an evolutionarily conservedATP-dependent chromatin-remodeling com-plex SWI/SNF, was discovered in our labora-tory. We showed that the first 38 amino acidsof Swi1 were able to aggregate, and maintainand propagate [SWIC]. However, further dele-tion to the first 32 amino acids resulted in a dra-matic reduction in aggregation, indicating that


the minimal prion domain (PrD) of Swi1 liesbetween residues 32 to 38. Further analysisshowed that the first 33 amino acids of Swi1are able to aggregate, and maintain a prion con-formation in the absence of full-length Swi1,suggesting that this region is likely the minimalPrD of Swi1. Using a newly designed reportersystem that can faithfully report the prion statusof Swi1, we conducted high-throughput screensto identify compounds that can eliminate orinhibit [SWIC] and have obtained a number ofpromising anti-[SWIC] compounds. We are cur-rently elucidating the hit compounds’ mecha-nism of action and investigating their ability toantagonize PrPSc and inhibit Ab-induced toxic-ity in a mammalian cell culture system. Thesestudies will shed light on the mechanisms ofprotein misfolding, aggregation, and amyloidfiber formation - all of which are relevant toprion diseases and other amyloid-based neuro-logical disorders.

P.146: Studying b-helical PrPSc

constructs in silico

Lyudmyla Dorosh1,3, Holger Wille2,4, andMaria Stepanova1,3

1Department of Electrical and Computer

Engineering; University of Alberta; Edmonton,

Alberta, Canada; 2Centre Prions and Protein

Folding Diseases; Edmonton, Alberta, Canada;3National Institute for Nanotechnology; Edmonton,

Alberta, Canada; 4Department of Biochemistry;

University of Alberta; Edmonton, Alberta, Canada

Misfolding and aggregation of prion proteins(PrP) are believed to cause the transmissiblespongiform encephalopathies - lethal neurode-generative diseases in mammals. The 3D struc-ture of the infectious isoform, PrPSc, and thepathways of conversion remain largelyunknown. We report our attempts to buildb-rich aggregates from a bovine PrP106 pep-tide as well as from a full-length bovine PrPconstruct using a combination of homology anddocking modeling. For this purpose we usepublished threading data, as well as built andaligned several novel threads. For stability

evaluations of the modeled systems, multiplems-long all-atom molecular dynamics (MD)simulations in explicit solvent have been per-formed using the Gromacs package. The struc-ture evolution, hydrogen bonding, and essentialcollective dynamics trends have been analyzedfrom the MD data. It was shown that most ofthe smallest stable threaded units comprise 2b-helical rungs, whereas an inclusion of thethird rung does not satisfy the threading rules.Modeling of b-helices from the PrP106 peptideresulted in stable N-term-N-term right-handedb-helical (RH) octamer complexes slightlytwisted along the fibril axis and less stable N-term-C-term-C-term-N-term RH complexes.Both RH and left-handed (LH) C-term-C-termcomplexes maintained the aggregated structure,but exhibited a low b content. However, C-ter-minus based complexes formed more hydrogenbonds than N-terminus based complexes.

P.147: Small molecule inhibitors ofmutant PrPC toxicity and

PrPsc formation

James Heeres1, Thibaut Imberdis1,Han Yueh2, Aaron Beeler2, and

David Harris

1Boston University School of Medicine; Boston,

MA, USA; 2Boston University; Boston, MA, USA

Since the function of cellular prion protein(PrPC) is still poorly understood, discoveringcompounds that target these functions has beendifficult, with candidate compounds typicallyidentified based on their ability to disrupt PrPscinfection/proliferation in cell culture. Ourobjective is to develop compounds capable ofdisrupting functionally important PrPC-relatedpathways. Our laboratory has previouslyreported on a central region deletion of PrPC(DCR) whose expression results in severalinteresting phenotypes, including 1) selectedantibiotic hypersensitivity (DBCA, [1,2]), 2)induction of spontaneous ionic currents[3,4],and 3) acute neuronal toxicity in transgenicmice[5]. We undertook a high-throughputscreen to identify inhibitors of DCR-induced


antibiotic hypersensitivity, and analyzed theirmodulation of PrPC-related processes. Severalclasses of small molecule leads were identified,one of which, designated LD49, we report onhere. LD49 potently suppress DCR-inducedantibiotic hypersensitivity in the DBCA, andalso inhibits PrPsc production in prion-infectedN2a cells. Interestingly, LD49 shows structuralsimilarity to published ligands[6–8] for a-2-delta, an auxiliary subunit of voltage-gated cal-cium channels, and known PrPC interactor[9],thus raising the possibility that a-2-delta is thetarget for LD49. However, we have observedonly partial correlation between the a-2-deltabinding activity of LD49 analogs from the liter-ature and their activity in the DBCA. Effortsare underway to test the role of a-2-delta in theaction of LD49, as well as identifying its targetusing unbiased strategies. We hope to useLD49 and other lead molecules from our screento elucidate PrPC-related neurotoxic pathways,and to further develop these pharmacophoresfor therapeutic intervention.

P.148: Key steric zipper segments governconversion by mouse and elk prions

Timothy D Kurt1, Lin Jiang2, Hasier Erana3,Jun Liu1, Joaqu�ın Castilla3,4,

David Eisenberg2, andChristina J Sigurdson1,5

1Departments of Pathology and Medicine; UC San

Diego; La Jolla, CA USA; 2UCLA-DOE Institute;

Howard Hughes Medical Institute; and Molecular

Biology Institute; UCLA; Los Angeles, CA USA;3IKERBASQUE; Basque Foundation for Science;

Bilbao, Spain; 4CIC bioGUNE; Parque Tecnol�ogicode Bizkaia; Derio, Spain; 5Department of

Pathology, Immunology, and Microbiology; UC

Davis; Davis, CA USA

The molecular mechanism by which PrPC isconverted to PrPSc remains poorly understood,yet is clearly influenced by (1) the conforma-tion of the PrPSc and (2) PrPC and PrPSc

sequence similarity. Sequence complementarityis also an important determinant of seeding in

other amyloid proteins, such as amyloid-b,a-synuclein and tau.

How do specific amino acid side chainsinfluence prion conversion? Eisenberg and col-leagues have shown that the atomic-level struc-ture of amyloid fibrils formed by peptides fromPrP, amyloid-b, tau and other amyloidogenicproteins consists of a repetitive motif: b-sheetsarranged with self-complementary interdigitat-ing amino acid side chains at the interface.

We hypothesize that sequence complemen-tarity within key short segments of PrP impactsprion conversion across many strains and spe-cies. In support of this hypothesis, we havefound that amino acid substitutions associatedwith conformational changes within the b2-a2 loop, or with long-distance interactionsbetween the loop and the third helix, do notimpact conversion. Instead, seeded prion con-version appears to be controlled by amino acidsequence within key segments of the host PrPC.Interestingly, PrPC variants with substitutionsin certain segments are efficiently converted bysome mouse prion strains but not others, sug-gesting that the steric zippers involved in prionconversion may vary by PrPSc conformation.

This work provides important insights intospecies barriers to prion transmission as wellthe molecular basis for self-templating amyloidformation.

P.149: Synthetic amyloid fibrilsgenerated from the N-terminal prion

protein fragment 23–144 causetransmissible prion disease in mice

Jin-Kyu Choi1, Ignazio Cali2,Krystyna Surewicz1, Qingzhong Kong2,

Pierluigi Gambetti2, andWitold K Surewicz1

1Department of Physiology and Biophysics; School

of Medicine; Case Western Reserve University;

Cleveland, OH USA; 2Department of Pathology;

School of Medicine; Case Western Reserve

University; Cleveland, OH USA

The Y145Stop mutation in PRNP, resultingin the C-terminally truncated prion protein


PrP23–144, is associated with a familial, GSS-like prion disease with extensive PrP-amyloiddeposits. Even though previous attempt totransmit this human disease to rodents has notbeen successful, here we report that Tga20mice inoculated with amyloid fibrils generatedfrom the recombinant mouse PrP23–144 devel-oped clinical symptoms of prion disease with100% attack rate. The incubation period in thefirst passage was 254§ 12 days; it was reducedto »210 d in subsequent passages. Neuropatho-logical examination showed severe spongiformdegeneration and accumulation of PrP plaques,and Western blot analysis revealed the presenceof proteinase K (PK)-resistant PrP similar tothat observed in classical strains of scrapie(»25–30 kDa; »19 kDa upon deglycosylation)as well as smaller PK-resistant fragments of 5–6 and 8–9 kDa. The latter fragments appear tobe remarkably similar to those observed in GSSphenotypes. moPrP23–144 amyloid fibrils werealso infectious to wild-type mice. Furthermore,these fibrils were effective as a seed for theconversion of brain PrPC using serial PMCAprotocol in vitro, producing a PK-resistantproduct similar to that generated in vivo. Thefinding that synthetic PrP23–144 fibrils arebona fide prions have important implicationsfor understanding mechanistic and structuralaspects of prion protein conversion, suggestingthat seeded prion protein conversion to PrPSc

state is initiated by element(s) within the»110–140 segment (which correspond to theb-core of PrP fibrils as defined by solid-stateNMR), from which PK-resistant b-structurepropagates into the surrounding regions.

P.150: Assessing the pathogenicity ofrare PRNP variants by comparing case

and control allele frequency

Eric Vallabh Minikel1,2,3,4,Sonia M Vallabh1,2,4, Monkol Lek2,3,

Karol O Estrada2,3, Kaitlin E Samocha2,3,4,J Fah Sathirapongsasuti5, Cory Y McLean5,

Joyce Y Tung5, Linda PC Yu5,Pierluigi Gambetti6, Janis Blevins6,Shulin Zhang7, Yvonne Cohen6,Wei Chen6, Masahito Yamada8,

Tsuyoshi Hamaguchi8, Nobuo Sanjo9,Hidehiro Mizusawa10,Yosikazu Nakamura11,

Tetsuyuki Kitamoto12, Steven J Collins13,Alison Boyd13, Robert G Will14,

Richard Knight14, Claudia Ponto15,Inga Zerr15, Theo FJ Kraus16,

Sabina Eigenbrod16, Armin Giese16,Miguel Calero17, Jes�us de Pedro-Cuesta17,

St�ephane Ha€ık18,19,Jean-Louis Laplanche20,Elodie Bouaziz-Amar20,

Jean-Philippe Brandel18,19,Sabina Capellari21,22, Piero Parchi21,22,

Anne H O’Donnell-Luria2,3,23,Konrad J Karczewski2,3,

Jamie L Marshall2,3, Michael Boehnke24,Markku Laakso25, Karen L Mohlke26,Anna K€ahler27, Kimberly Chambert28,

Steven McCarroll28, Patrick F Sullivan26,27,Christina M Hultman27, Shaun M Purcell29,

Pamela Sklar29, Sven J van der Lee30,Annemieke Rozemuller31, Casper Jansen31,

Albert Hofman30, Robert Kraaij32,Jeroen GJ van Rooij32, M Arfan Ikram30,

Andr�e G Uitterlinden30,32,Cornelia M van Duijn30,

Exome Aggregation Consortium (ExAC)33,Mark J Daly2,3, and Daniel G MacArthur2,3


1Prion Alliance; Cambridge, MA USA; 2Program in

Medical and Population Genetics; Broad Institute;

Cambridge, MA USA; 3Analytical and Translational

Genetics Unit; Massachusetts General Hospital;

Boston, MA USA; 4Program in Biological and

Biomedical Sciences; Harvard Medical School;

Boston, MA USA; 5Research; 23andMe Inc.;

Mountain View, CA USA; 6National Prion Disease

Pathology Surveillance Center; Cleveland, OH

USA; 7University Hospitals Case Medical Center;

Cleveland, OH USA; 8Department of Neurology and

Neurobiology of Aging; Kanazawa University

Graduate School of Medical Sciences; Kanazawa,

Japan; 9Department of Neurology and Neurological

Science; Graduate School; Tokyo Medical and

Dental University; Tokyo, Japan; 10National Center

Hospital; National Center of Neurology and

Psychiatry; Tokyo, Japan; 11Department of Public

Health; Jichi Medical University; Shimotsuke,

Japan; 12Department of Neurological Science;

Tohoku University Graduate School of Medicine;

Sendai, Japan; 13Australian National Creutzfeldt-

Jakob Disease Registry; The University of

Melbourne; Parkville, Australia; 14National

Creutzfeldt-Jakob Disease Research and

Surveillance Unit; Western General Hospital;

Edinburgh UK; 15National Reference Center for

TSE; Georg-August University; Goettingen,

Germany; 16Center for Neuropathology and Prion

Research (ZNP) at the Ludwig-Maximilians-

University; Munich, Germany; 17Instituto de Salud

Carlos III and CIBERNED; Madrid, Spain;18Inserm U 1127; CNRS UMR 7225; Sorbonne

Universit�es; UPMC Univ. Paris 06 UMR S 1127;

Institut du Cerveau et de la Moelle �epini�ere; ICM;

Paris, France; 19AP-HP; Cellule Nationale de

R�ef�erence des Maladies de Creutzfeldt-Jakob;

Groupe Hospitalier Piti�e-Salpetri�ere; Paris,France; 20Service de Biochimie et Biologie

mol�eculaire; Hopital Lariboisi�ere; Paris, France;21IRCCS Institute of Neurological Sciences;

Bologna, Italy; 22Department of Biomedical and

Neuromotor Sciences; University of Bologna;

Bologna, Italy; 23Division of Genetics and Geno-

mics; Boston Children’s Hospital; Boston, MA

USA; 24University of Michigan School of Public

Health; Ann Arbor, MI USA; 25Department of Med-

icine; University of Eastern Finland and Kuopio

University Hospital; Kuopio, Finland; 26Depart-

ment of Genetics; University of North Carolina

School of Medicine; Chapel Hill, NC USA;

27Karolinska Institutet; Stockholm, Sweden; 28Stan-

ley Center for Psychiatric Research; Broad Insti-

tute; Cambridge, MA USA; 29Icahn School of

Medicine at Mount Sinai; New York, NY USA;30Department of Epidemiology; Erasmus MC; Rot-

terdam, The Netherlands; 31Prion Surveillance

Center; Department of Pathology; University Medi-

cal Center; Utrecht, Netherlands; 32Department of

Internal Medicine; Erasmus MC; Rotterdam, The

Netherlands; 33A list of consortium members may be

found at http://exac.broadinstitute.org/about

To date, 64 different genetic variants in thePRNP gene have been reported to cause priondisease in humans.1The majority of these havebeen observed in only one or a few patients,and pervasive ascertainment bias, low rates ofpredictive genetic testing and frequent lack offamily history2–5make it impossible to evaluatethe penetrance of the vast majority of variants.Here we systematically assess the disease pene-trance of PRNP variants by comparing allelefrequencies in >13,000 prion disease casesreported to surveillance centers in 8 countriesagainst allele frequencies in >60,000 controlindividuals sequenced by the Exome Aggrega-tion Consortium and »500,000 individualsgenotyped by 23 and Me. Reportedly patho-genic variants occur in controls »100 timesmore frequently than expected under anassumption of complete penetrance, given theknown disease incidence. Variants with thestrongest prior evidence of pathogenicity areindeed very rare, consistent with high pene-trance. Other variants are enriched by »4-foldto »250-fold in cases over controls, indicatingthat these variants dramatically increase priondisease risk but still yield low lifetime pene-trance. Still other reportedly pathogenic var-iants are reasonably common in controlpopulations, and may confer little or no diseaserisk. Heterozygous nonsense and frameshiftvariants truncating the prion protein in its N ter-minus occur in healthy control individuals, incontrast to C-terminal truncating variants foundin prion disease patients, indicating that a 50%reduction in gene dosage for PRNP is well-tol-erated in humans and supporting the safety oftherapeutic reduction of prion proteinexpression.


P.151: Copper complexation of smallpeptides modeling the fundamental

chemistry of copper complexation to theprion protein

Cheryle Beuning*, Estela Maganalles,Irma Sanchez Lombardo, Donn Calkins,

and Debbie Crans

Chemistry Department; Colorado State University;

Fort Collins, CO USA

Metal complexation to prion proteins mayimplicated in the pathway of aggregation of theprion protein as has been reported for the amy-loid-b proteins resulting in insoluble plaquesthat deposit in the brain and lead to neurode-generation. In this presentation, we describeour use of small peptides to characterize themetal binding sites to and understand the bind-ing of copper to the model peptides and ulti-mately also the prion protein. The prionproteins bind metals through their amino andcarboxyl terminal groups, nitrogen or oxygenin the backbone, and certain side chains withnitrogen, sulfur, oxygen or that are charged.Common spectroscopic techniques can be usedto study these metal binding areas includingnuclear magnetic resonance (NMR) and massspectrometry (MS). 1H and 13C NMR spectros-copy were used to monitor metal ion complexa-tion. 1H NMR spectra allows observation ofcomplexation and 13C NMR allows observationof the binding sites by monitoring changes inthe intensity of chemical signals. Complexesformed upon addition of copper salts to peptidesolutions will also be identified by electrosprayionization MS. Lastly, reverse micelles will beemployed along with NMR techniques to char-acterize the complexes formed in small inho-mogeneous environments like those found inthe brain between neurons. If one understandshow the metal forms complexes with the prionprotein one can develop the knowledge to beused to design compounds that inhibit this com-plexation and begin to probe the role of themetal ion complexation.

P.152: Relationship of PrPSc molecularproperties with incubation time in a

natural prion disease host: Acharacterization of 3 isolates of US

sheep scrapie

Catherine Vrentas1,2,*, Jodi Smith1,3,Justin Greenlee1, and Eric Nicholson1

1Agricultural Research Service; US Department of

Agriculture; Ames, IA USA; 2Frostburg State

University; Frostburg, MD USA; 3Iowa State

University; Ames, IA USA

Determination of aspects of tertiary and qua-ternary structure of PrPSc associated with dif-ferences in disease presentation in the host is akey area of interest in the prion field. Previ-ously, we determined that a US scrapie isolate(136-VDEP) with a short incubation time uponpassage in sheep also exhibited low PrPSc sta-bility in guanidine hydrochloride (GdnHCl), ascompared to 2 isolates with longer incubationtimes and higher GdnHCl stability (136-A and13–7). Here, we utilize this natural host systemfor a more in-depth examination of PrPSc bio-chemical properties. First, we recapitulated theincubation time findings from sheep in ovinized(VV136) transgenic mice via intracranial inoc-ulation of 136-VDEP and 136-A. In contrast topublished results in rodent strains, lowerGdnHCl stability of 13-VDEP from sheep braindid not correlate with decreased stability ofaggregates in the presence of SDS and heat.While the aggregate stability assay involvesproteinase K (PK) treatment, the lack of corre-lation cannot be explained by a separate PK-sensitive population of PrPSc, since all 3 iso-lates exhibit similar PK sensitivity profiles,without a large fraction of PK-sensitive mate-rial. However, a time course of GdnHCl treat-ment suggests the presence of 2 distinctpopulations of PrPSc in the brain homogenates.Since oligomeric aggregates have been associ-ated with higher infectivity, we examinedPrPSc size distributions by sucrose gradientfractionation, but did not observe differences


between isolates. We suggest that in this sys-tem, phenotypical differences may be due totertiary structural, as opposed to quaternarystructural, differences.

P.153: An independent and blindedconfirmation of real-time quaking-

induced conversion (RT-QuIC) analysisof cervid rectal biopsies for detection of

chronic wasting disease

Sireesha Manne1,*, Naveen Kondru1,Nicholas Haley2, Tracy Nichols3,Bruce Thomsen4, Roger Main5,

Patrick Halbur5, Arthi Kanthasamy1, andAnumantha Kanthasamy1

1Biomedical Sciences; Iowa State University; Ames,

IA USA; 2Kansas State University; Manhattan, KS

USA; 3United States Department of Agriculture;

Fort Collins, CO USA; 4National Veterinary

Service Laboratories; Ames, IA USA; 5VDPAM;

Iowa State University; Ames, IA USA

Prion diseases are transmissible spongi-form encephalopathies (TSEs) characterizedby an always fatal, progressive neuronaldegeneration in the brain due to infectiousmisfolded prion proteins whose prolongedincubation periods often make ante-mortemdiagnosis difficult. Chronic wasting disease(CWD) is a TSE affecting both wild andcaptive populations of mule deer, white-tailed deer, elk and moose. CWD in cervidswas first identified in Rocky Mountain Statesand has recently spread to several otherstates including Iowa. In this current study,we attempted to independently confirm theresults of a Real-Time Quaking-InducedConversion (RT-QuIC) assay to diagnoseCWD using rectal biopsy sections fromfarmed white-tailed deer. First, we generatedrecombinant prion protein substrate and thenvalidated the quality of protein for RT-QuICusing a reference prion protein kindly pro-vided by Dr. Caughey’s lab. After validatingthe assay, we blindly evaluated approxi-mately 350 rectal biopsy samples analyzedpreviously by another institution. All assay

plates included positive and negative controlsand were analyzed in triplicate. Sampleswere analyzed using the Biotek Cytation-3multimode plate reader for 24-hrs duration.Our RT-QuIC assays showed 55% positivityfor 356 rectal samples analyzed. Comparisonof RT-QuIC results with the immunohisto-chemical results of obex revealed 93% sensi-tivity (95% confidence limits: 88.05–95.78%)and 96% specificity (95% CL: 91–99%),confirming that the RT-QuIC assay may beone of the most promising rapid assays fordetecting CWD prions. We are currentlyworking on applying the RT-QuIC assay toother test samples (ISU Presidential Wildlifeinitiative, ISU-CVM Diagnostic lab andES10586).

P.154: Brain derived lipids inhibit prionamyloid formation in vitro

Clare Hoover, Davin Henderson,Mark Zabel, and Edward Hoover


The normal cellular prion protein (PrPC)resides in cellular outer membrane lipid raftsand conversion from PrPC to the pathogenicmisfolded isoform is believed to occur at thelipid membrane. In vitro assays have demon-strated the intimate association between prionconversion and lipids, specifically phosphati-dylethanolamine, which is a critical cofactor inthe formation of synthetic infectious prions. Inthe current work, we demonstrate an opposingproperty of lipids, the ability to inhibit amyloidformation in vitro. The real-time quaking-induced conversion assay (RT-QuIC) was usedto investigate whole brain lipid effects on prionamyloid formation. An alcohol based extractiontechnique was used to remove the lipid contentfrom terminal chronic wasting disease (CWD)-infected white tailed deer brain homogenates.Eliminating lipids increased the sensitivity ofRT-QuIC detection of CWD in brain samplesone hundred-fold. Addition of brain-derivedlipid extracts to CWD prion samples inhibitedamyloid formation in a dose-dependent


manner. Brain-derived lipids also inhibitedprion amyloid formation in RT-QuIC reactionseeds derived from lymphoid tissues. This isthe first demonstration of brain derived lipidsdirectly inhibiting prion amyloid formation invitro and highlights the diverse roles lipids playin the conversion process. Further experimentswill identify the individual lipid species orgroups of lipids responsible for this inhibitoryactivity.

P.155: Quantitative real-time analysis ofdisease specific tau amyloid

seeding activity

Davin Henderson and Edward Hoover

Prion Research Center; College of Veterinary

Medicine and Biomedical Sciences; Colorado State

University; Fort Collins, CO USA

A leading hypothesis for the cause of neuro-degenerative diseases is the templated misfold-ing of cellular proteins to an amyloid state.Spongiform encephalopathies were the first dis-eases discovered to be caused by a misfoldedamyloid-rich protein. It is now recognized thatthe major human neurodegenerative diseases,including Alzheimer’s disease (AD),Parkinson’s disease (PD), and chronic trau-matic encephalopathy (CTE), also are associ-ated with amyloid formation. Moreover, ADand PD amyloids have been shown competentto transmit disease in experimental animalmodels, suggesting shared mechanisms withtraditional prion diseases. Sensitive detectionof prion disease has been advanced by in vitroamplification of low levels of disease specificamyloid seeds, e.g. serial protein misfoldingamplification (sPMCA), amyloid seeding(ASA) and real-time quaking induced conver-sion (RT-QuIC), thereby replicating the diseaseprocess in vitro. In addition, measurement ofthe amyloid formation rate can estimate thelevel of disease-associated seed by using meth-ods analogous to quantitative polymerase chainreaction (qPCR). In the present work, we applythese principles to show that seeding activity ofin vitro generated amyloid tau and AD brain

amyloid tau can be readily detected andquantitated.

P.156: The cellular form of the prionprotein PrPC is processed to varying

degrees in different species andPRNP genotypes

Wilfred Goldmann1, Paula Stewart1,Chloe Brown1, Kayleigh Iremonger1,

Boon Chin Tan1, Dorothy Kisielewski1,Lauren Laing1, Candace Mathiason3,Francesca Chianini2, Fiona Houston1,Jean Manson1, and Michael Tranulis4

1The Roslin Institute; R(D)SVS University of

Edinburgh; Easter Bush , near Edinburgh, Scotland,

UK; 2The Moredon Institute; Peniciuk, near

Edinburgh, UK; 3Prion Research Center; Colorado

State University; Fort Collins, CO USA;4Norwegian School of Veterinary Science;

Oslo, Norway

Cellular prion protein PrPC may be describedas a proprotein, in which the active peptides arereleased by proteolytic cleavage at variouspositions, e.g. a-cleavage (ovine codon 114) orC-terminal cleavage (ovine codon 231). By thisshedding process different PrPC domains willbe released, such as PrP25-114 (also calledN1), PrP25-231 or retained on the cell surface,such as PrP115-234 (C1), and full lengthPrP25-234. The in vivo function of these PrPC

fragments and the proteases involved aremostly unresolved, but there is no doubt thatthese fragments can make up significantamounts of total PrPC levels in mammalianbrain tissue. We have previously shown anassociation between ovine PRNP genotypesand the level of cleavage products in brain sug-gesting a link with scrapie susceptibility. Wehave analyzed the steady-state levels of full-length PrPC, C1 and C2 in brain material froma number of rodent, carnivore and ruminantspecies. We conclude that there are consider-able differences in the ratio of PrPC to C1 andthe presence of C2 among mammalian species.For example gray squirrel cortex had 10% C1


of total PrPCwhich was the lowest of all sam-ples analyzed, whereas sheep of the ARR/ARRgenotype exhibited 5 times more C1, on aver-age 53% of total PrPC. Increased total PrPC

expression is associated with the relative levelof truncated forms. It is likely that these differ-ences in PrPC processing contribute to the sus-ceptibility and pathogenesis of prion diseasesand they may reflect on diverse biological rolesin different species.

P.157: Uptake of prions into plants

Christopher Johnson1, Christina Carlson1,Matthew Keating1,2, Nicole Gibbs1,Haeyoon Chang1, Jamie Wiepz1, and

Joel Pedersen1

1USGS National Wildlife Health Center; Madison,

WI USA; 2University of Wisconsin - Madison;

Madison, WI USA

Soil may preserve chronic wasting disease(CWD) and scrapie infectivity in the environ-ment, making consumption or inhalation of soilparticles a plausible mechanism whereby na€ıveanimals can be exposed to prions. Plants areknown to absorb a variety of substances fromsoil, including whole proteins, yet the potentialfor plants to take up abnormal prion protein(PrPTSE) and preserve prion infectivity is notknown. In this study, we assessed PrPTSE

uptake into roots using laser scanning confocalmicroscopy with fluorescently tagged PrPTSE

and we used serial protein misfolding cyclicamplification (sPMCA) and detect and quantifyPrPTSE levels in plant aerial tissues. Fluores-cence was identified in the root hairs of themodel plant Arabidopsis thaliana, as well asthe crop plants alfalfa (Medicago sativa), bar-ley (Hordeum vulgare) and tomato (Solanumlycopersicum) upon exposure to tagged PrPTSE

but not a tagged control preparation. UsingsPMCA, we found evidence of PrPTSE inaerial tissues of A. thaliana, alfalfa and maize(Zea mays) grown in hydroponic cultures inwhich only roots were exposed to PrPTSE. Lev-els of PrPTSE in plant aerial tissues rangedfrom approximately 4 £ 10¡10 to 1 £ 10¡9 g

PrPTSE�g¡1 plant dry weight or 2 £ 105 to7 £ 106 intracerebral ID50 units�g¡1 plant dryweight. Both stems and leaves of A. thalianagrown in culture media containing prions areinfectious when intracerebrally-injected intomice. Our results suggest that prions can betaken up by plants and that contaminated plantsmay represent a previously unrecognized riskof human, domestic species and wildlife expo-sure to prions.

P.158: Evaluation of prion vaccineadministered with vaccine enhancing

agent

Valerie Johnson, Steve Dow, andMark Zabel

Colorado State University; Fort Collins; CO USA

Transmissible spongiform encephalopathy(TSE) is a neurodegenerative disorder charac-terized by pathologic accumulation of a mis-folded form of a normal cellular protein inneurons. Emergence of TSEs in wildlife popu-lations and the ability of some TSEs to crossspecies barriers have prompted concern regard-ing the lack of treatment options or preventionstrategies. Efforts at vaccine development havebeen hampered by the difficulty of overcomingself-tolerance. Studies in our lab have demon-strated that vaccine induced immunity is oftendiminished due to the recruitment of anti-inflammatory myeloid cells. We hypothesizedthat utilizing an effective antigen while inhibit-ing monocyte migration could elicit a moreeffective anti-prion response.

The vaccine was formulated using a peptidefragment of the human prion protein (PrP106-126). This peptide spontaneously forms fibrillaraggregates and is thought to mediate the con-version from the normal cellular prion protein(PrPC) to the pathogenic form (PrPSC). Toenhance vaccine efficacy, a monocyte migra-tion inhibitor was administered (RS102895).To further target the pathogenic PrPSC, thepeptide was reconstituted in an acidic solutionand incubated at 37�C to increase fibrillization.Antibody responses were assessed using


ELISA and Western Blot. Vaccinated miceexhibited increased antibody titers in additionto a cell mediated immune response. Micetreated with RS 102895 also exhibitedincreased concentrations of antibodies againstboth PrP 106–126 and PrPC.

This vaccination regime shows great prom-ise in eliciting an immune response, thus over-coming self-tolerance. Our results suggest thatthis strategy could overcome the limitationsthat have prevented successful development ofa prion vaccine.

P.159: Improvements of nasal brushingprocedure for Creutzfeldt-Jakob disease

diagnosis

Matilde Bongianni1, Christina Orr�u2,Giovanni Tonoli3, Bradley Groveman2,Giorgo Triva4, Santina Castriciano4,Luca Sacchetto1, Andrew Hughson2,Annachiara Cagnin5, Stefano Capaldi1,

Sergio Ferrari1, Michele Fiorini1,Salvatore Monaco1, Maurizio Pocchiari6,Byron Caughey2, and Gianluigi Zanusso1

1University of Verona; Verona, Italy; 2Rocky

Montain Laboratories; Hamilton, MT USA;3Ospedale “Santa Maria della Misericordia”;

Rovigo, Italy; 4Copan Italia S.P.A.; Brescia, Italy;5University of Padua; Padua, Italy; 6Istituto

Superiore di Sanit�a; Rome, Italy

Introduction. We previously identified prionseeding activity in olfactory mucosa (OM) ofCJD patients using nasal brushings coupledwith Real Time Quaking induced Conversion(RT-QuIC) with 100% specificity and >97%sensitivity. OM samples were collected using asterile disposable Cyto-brush (Kito-Brush, Kal-tek) which might provoke a mild discomfortfor patients. Therefore, we aimed to use a moregentle tool for OM samplings such as shortnylon fiber Flocked swabs (Copantechnologies).

Materials and Methods. We collected OMand CSF samples in 43 CJD patients. To ensure

efficient OM sample collection, each patientunderwent to two OM samplings using flockedswabs one in each nostril and a final with Cyto-brush. OM samples were processed and ana-lyzed by RT-QuIC, as previously described.

Results. Using Cyto-brushes 32 out of 35 OMsamples were positive by RT-QuIC analysis,while flocked swabs in 40 out of 43 OM sam-ples. In contrast, CSF samples were positive in33 out of 43. Two OM samples resulted nega-tive for both Cyto-brush and Flocked swab.Neither OM sampling technique or CSF pro-duced false positives.

Conclusion. This study demonstrates thatOM brushing following RT-QuIC ssay is 95%sensitive and 100% specific in CJD diagnosiswhile CSF resulted 77% sensitive. OM collec-tion using flocked swabs is preferred and pro-vides same sensitivity as cyto-brush. Thesedata recommend four separate samplings possi-bly from both nostrils to maximize the sensitiv-ity, using three Flocked swabs and lastly abrush.

P.160: Detecting the temporal status ofprionemia in transmissible spongiform

encephalopathy-infected hosts

Alan Elder1, Davin Henderson1,Amy Nalls1, Kristen Davenport1,

Anthony Kincaid2, Edward Hoover1,Jason Bartz2, and Candace Mathiason1

1Colorado State University; Fort Collins, CO USA;2Creighton University; Omaha, NE USA

Infectious prions can traverse epithelial bar-riers and gain access to the circulatory systemearly in infection. The details of prion entry,temporal status, and persistence in the bloodremain unknown. Furthermore, it is unknown ifthe route of inoculation plays a role in thedevelopment of prionemia. We previouslydemonstrated PrPC amyloid conversion activityin the blood (prionemia) of deer and hamstersinfected with transmissible spongiform enceph-alopathies (TSEs) using whole blood real-time


quaking-induced conversion (wbRT-QuIC). Inthis study we analyzed the temporal status ofprionemia, spanning 0–100% of the diseasecourse, in hosts exposed to TSEs via bloodtransfusion or other peripheral means (i.e. oral,aerosol, extranasal, and subcutaneous). Ourresults demonstrate the presence of PrPC

amyolid conversion activity in the blood of allTSE-inoculated hosts as early as 15 minutespost inoculation likely-representing the pointsource inoculum–which was cleared from thecirculatory system by 72 hours post exposure.De novo host generated hematogenous PrPC

amyloid conversion activity, or prionemia, wasidentified at 4–5% of the TSE disease courseand persisted throughout disease. Our resultsindicate that hematogenous prions can traversemucosal surfaces and enter the circulatory sys-tem with the same speed and efficiency as thoseentering the blood directly by blood transfu-sion, and that an asymptomatic carrier state isestablished within minutes of TSE exposure.

P.161: Prion soil binding may explainefficient horizontal CWD transmission

Nathaniel Denkers1, Davin Henderson1,Shannon Bartelt-Hunt2, Jason Bartz3, and

Edward Hoover1

1Colorado State University; Fort Collins, Colorado

USA; 2University of Nebraska-Lincoln; Omaha,

Nebraska USA; 3Creighton University; Omaha,

Nebraska USA

Background. Chronic wasting disease(CWD) is unique due to the facile spread innature. The interaction of excreted CWD prionsand soil is a hypothesized contributor in envi-ronmental transmission. The present studyexamines whether and to what degree CWDprions bind to silty clay loam (SCL) using anadapted version of real-time quaking-inducedconversion (RT-QuIC) methodology.

Materials and Methods. Varying amounts(50–3.12 mg) of SCL were incubated with1 mL-serial dilutions of CWD (C), CWD (¡),

or no brain homogenate (BH). Samples werecentrifuged, washed, diluted 1:10 in 0.1% SDS,and 2.5 uL seeded in RT-QuIC assays employ-ing recombinant Syrian hamster prion PrP sub-strate. Multiple well replicates of sample andsupernatant fractions were assayed for positiveseeding activity (recorded as thioflavin T fluo-rescence emission; 480 nm). Samples wereconsidered positive if they crossed a thresholdof 25,000. Reaction rates (RR) were calculated,averaged, and expressed as 1/RR.

Results. Positive seeding activity wasdetected for most SCL samples incubatedwith CWD (C) BH dilutions. Higher SCLconcentrations (50 mg) produced low fluores-cent readings due to optical interference.Lower SCL concentrations (6.25 mg) pro-duced minimal optical interference andremoved the vast majority of seeding activityfrom CWDC BH in a concentration-depen-dent manner; determined by seeding activityin residual BH supernatants. Control SCLand supernatants produced minimal false-positive reactions (8 of 240 replicates;3.3%). We estimated the prion bindingcapacity of SCL to be 0.16 ng/mg.

Conclusion. Silty clay loam exhibits highlyefficient prion binding, inferring a durableenvironmental reservoir, and an efficientmechanism for indirect horizontal CWDtransmission.

P.162: Prion protein cleavage fragmentsmodulate neural stem cellrenewal and migration

Cathryn Haigh and Steven Collins

The University of Melbourne; Victoria, Australia

Neural stem cells (NSCs) are now recog-nized to persist in the brain into old age andpossibly throughout life. They present a poten-tial new avenue of therapeutic targeting in neu-rodegenerative diseases and aging.Understanding the factors that influence theirgrowth will be essential for any therapies that


target these cells. The normal function of theprion protein (PrP) has remained elusive. PrPundergoes at least 2 internal cleavage events toproduce N1/C1 and N2/C2 fragments. We haveproposed that these distinct fragments possessdiffering properties and physiological function.Our previous studies have shown that the N-ter-minal cleavage fragment designated N2reduces the production of intracellular reactiveoxygen species (ROS) in response to mildstress. Other research groups have shown pro-tective effects of N1. NSC growth is modulatedby intracellular ROS levels and NSCs harvestedfrom mice expressing different levels of PrPshow a positive correlation between PrPexpression and growth. We hypothesized thatthe N2 fragment and also the longer N1 frag-ment might be able to modulate NSC growththrough their effects on modulating intracellu-lar ROS. We find that both the N1 and N2 frag-ments halt cellular growth, migration andmaturation. NSCs show reduced intracellularROS detection following N1 or N2 exposureand appear to have entered into a quiescentstate. Inhibition of NADPH oxidase produces asimilar phenotype in these cells. Our investiga-tions now focus on the role of N1 and N2 mod-ulation of NADPH oxidase signaling pathwaysin maintaining stem cell quiescence.

P.163: A practical approach to avoidingiatrogenic CJD frominvasive instruments

Paul Brown1 and Michael Farrell2

1NIH (retired); Bethesda, MD USA; 2Beaumont

Hospital; Dublin, Ireland

Potential Creutzfeldt-Jakob disease instru-ment-contamination events continue to occurthat involve widespread hospital and patientconcern. This paper proposes a combination ofdiagnostic tests and instrument handling proce-dures that, if routinely applied to patientsadmitted with symptoms of either dementia orcerebellar disease, should eliminate the risk ofiatrogenic instrument infection.

P.164: Blood transmission of prioninfectivity in the squirrel monkey: The

Baxter study

Paul Brown1, Diane Ritchie2,James Ironside2, Christian Abee3,Thomas Kreil4, and Susan Gibson5

1NIH (retired); Bethesda, MD USA; 2University of

Edinburgh; Edinburgh, UK; 3University of Texas;

Bastrop, TX USA; 4Baxter Bioscience; Vienna,

Austria; 5University of South Alabama; Mobile,

AL USA

Five vCJD disease transmissions and an esti-mated 1 in 2000 ‘silent’ infections in UK resi-dents emphasize the continued need forinformation about disease risk in humans. Alarge study of blood component infectivity in anon-human primate model has now been com-pleted and analyzed. Among 1 GSS, 4 sCJD,and 3 vCJD cases, only GSS leukocytes trans-mitted disease within a 5–6 year surveillanceperiod. A transmission study in recipients ofmultiple whole blood transfusions during theincubation and clinical stages of sCJD andvCJD in ic-infected donor animals was uni-formly negative. These results, together withother laboratory studies in rodents and non-human primates and epidemiological observa-tions in humans, suggest that blood donationsfrom cases of GSS (and perhaps other familialforms of TSE) carry more risk than from vCJDcases, and that little or no risk is associatedwith sCJD. The issue of decades-long incuba-tion periods in ‘silent’ vCJD carriers remainsopen.


P.165: A prion protein-derived peptidereduces brain damage in an animal

model of stroke

Amanda Dossat, Efrosini Artikis,Katherine Wright, Caroline Strong,

Mohamed Kabbaj, and Ewa Bienkiewicz

Florida State University; Tallahassee, FL USA

The conformational duality of the prion pro-tein translates into strikingly distinct fates thisprotein can impart on the living systems. In onecase, the outcome is the irrevocable neurode-generation and fatal disease. In stark contrast,the cellular form of the prion protein (PrPc) par-takes in life-sustaining cellular functions thatinclude neuroprotection, signal transduction,and angiogenesis. Importantly, PrPc has beenshown to be involved in a natural response tovascular injury, with the cell damage and deathdue to stroke being significantly increased in its

absence. In vascular injury, including stroke,one of the key cell death-triggering events isthe release of toxic levels of free hemin. Apotential solution to this damage is a peptidetherapeutic agent that would neutralize hemin,thus reducing deleterious effects of bleeding.Both the hemorrhagic and ischemic stroke(with “microbleeds” inflicting secondary dam-age) would benefit from this approach. Usingbiophysical methods, we have identified a pep-tide derived from the PrPc N-terminus andtested a hypothesis that this hemin-sequesteringfragment could reduce brain damage due tobleeding. We employed a well-establishedmouse model of intracerebral hemorrhagicstroke (ICH) to test neurological/behavioraldeficiencies and brain tissue damage caused bystroke in the presence and absence of the pep-tide treatment. Our results indicate efficacy ofthe tested peptide at reducing deleteriouseffects of stroke in-vivo, making it a strong can-didate for further development as a novel thera-peutic intervention in vascular injury events.


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