the prion's elusive reason for being

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The Prions Elusive Reasonfor BeingAdriano Aguzzi, Frank Baumann, and Juliane BremerInstitute of Neuropathology, University of Zurich, CH-8091 Zurich, Switzerland,email: [email protected]

Annu. Rev. Neurosci. 2008. 31:43977

First published online as a Review in Advance onApril 2, 2008

The Annual Review of Neuroscience is online atneuro.annualreviews.org

This articles doi:10.1146/annurev.neuro.31.060407.125620

Copyright c 2008 by Annual Reviews.All rights reserved

0147-006X/08/0721-0439$20.00

Key Words

transmissible spongiform encephalopathy, prion diseases, PrPC, PrPSc

AbstractThe protein-only hypothesis posits that the infectious agent causingtransmissible spongiform encephalopathies consists of protein and lacksany informational nucleic acids. This agent, termed prion by StanleyPrusiner, is thought to consist partly of PrPSc, a conformational isoformof a normal cellular protein termed PrPC. Scientists and lay personshave been fascinated by the prion concept, and it has been subjectedto passionate critique and intense experimental scrutiny. As a result,PrPC and its isoforms rank among the most intensively studied proteinsencoded by the mammalian genome. Despite all this research, both thephysiological function of PrPC and the molecular pathways leading toneurodegeneration in prion disease remain unknown. Here we reviewthe salient traits of those diseases ascribed to improper behavior of theprion protein and highlight how the physiological functions of PrPC

may help explain the toxic phenotypes observed in prion disease.

439

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GPI: glycosylphos-phatidylinositol

Contents

TRANSMISSIBLE PRIONDISEASES ANDNONTRANSMISSIBLEPRION-RELATED DISEASES. . . . 440Prion Disease in Humans

and Animals . . . . . . . . . . . . . . . . . . . . 441The Nature of the Prion . . . . . . . . . . . 445Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . 447PHYSIOLOGICAL FUNCTION

OF THE CELLULAR PRIONPROTEIN . . . . . . . . . . . . . . . . . . . . . 447

Prion ProteinDecient Mice . . . . . . 447Functional Domains

of the Prion Protein . . . . . . . . . . . . . 449Point Mutations within

the Prion Protein . . . . . . . . . . . . . . . 451Evolution of the Prion Protein. . . . . . 452Cellular Processes Inuenced

by PrPC Expression . . . . . . . . . . . . . 452PrP and the Immune System . . . . . . . 457Molecular Mechanisms Mediating

the Function of PrPC . . . . . . . . . . . 457Interaction Partners of PrPC . . . . . . . . 461

CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 465

TRANSMISSIBLE PRIONDISEASES ANDNONTRANSMISSIBLEPRION-RELATED DISEASES

The origin of the word prion stems from theanagram of proteinaceous infectious particle.Naturally, the latter is by no means a qualify-ing attribute because all conventional infectiousagentsincluding all viruses and bacteriaareproteinaceous to some degree. What sets prionsapart, as proposed by Prusiner, is that the actualinfectious principle consists merely of proteinand is capable of replicating and transmittinginfections without the need for informationalnucleic acids. This postulate counters much ofthe established molecular biological evidence,which predicates that nucleic acids are the basisfor self-replicating biological information in all

living beings, including even the most elemen-tary infectious particles.

Prion diseases are generally characterizedby widespread neurodegeneration and there-fore exhibit clinical signs and symptoms of cog-nitive and motor dysfunction, in addition topropagating infectious prions and, in many in-stances, forming striking amyloid plaques. Thelatter plaques contain aggregates of PrPSc, amisfolded and beta-sheet-rich isoform of theprotein PrPC encoded by the PRNP gene. Fur-ther neuropathological features are neuronalloss, astrocytic activation (gliosis), and spongi-form change. All prion diseases are progressive,fatal, and presently incurable.

Although the normal cellular prion proteinPrPC can easily be digested with proteinase K(PK), the beta-sheet-rich, misfolded form PrPSc

is partially proteinase K (PK) resistant. A cru-cial piece of evidence demonstrating that PrPC

is a key player in prion disease came from ex-periments showing that mice lacking the prionprotein gene are resistant to prions (Bueler et al.1993).

Although the formation of PrPSc accompa-nies neurodegeneration in prion disease, manylines of evidence indicate that PrPSc is not in-trinsically neurotoxic. PrPC needs to be pre-sented by host neurons for neurodegenerationto occur. Thus, when neurografts propagat-ing PrPSc were implanted into Prnpo/o mice,the host mice did not develop prion disease(Brandner et al. 1996). Additionally, trans-genic mice expressing only a secreted form ofPrPC, lacking its membrane attachment via gly-cosylphosphatidylinositol (GPI) anchor, havebeen reported to be refractory to develop clini-cal signs of prion diseases, although prion inoc-ulation induces PrPSc formation and aggrega-tion of amyloid plaques (Chesebro et al. 2005).This nding indicates that membrane attach-ment of PrPC is a prerequisite for neurode-generation to occur and that the presence ofPrPSc alone does not cause disease. The im-portance of neuronal expression of PrPC forprion disease development has been corrobo-rated by the phenotype of mice with neuron-specic ablation of PrPC eight weeks after prion

440 Aguzzi Baumann Bremer

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inoculation. Early spongiform changes were re-versed, and clinical disease was prevented. Thisreversal occurred despite the accumulation ofextraneuronal PrPSc (Mallucci et al. 2003).

Bona de prion diseases are character-ized by their transmissibility and are there-fore also termed transmissible spongiform en-cephalopathies (TSE). Transmissibility is adening, and hence indispensable, trait of allprion diseases. However, transmissibility hasnot been formally proven for all kinds of dis-eases thought to be caused by prions. In ad-dition, some diseases are genetically associatedwith the prion protein, yet they are nontrans-missible. These diseases are sometimes calledprionopathies. Among these are rare geneticsyndromes that cosegregate with point muta-tions in the open reading frame of the PRNPgene. In addition to these naturally occurringprionopathies, several transgenic mice havebeen used to gain insight into functional do-mains of PrPC (Weissmann & Flechsig 2003).In these mice, deletion of parts of PrPC causedprionopathies, characterized by a shortened lifespan and the development of white matterdisease in the central nervous system (CNS)as well as neuronal cell death in the cere-bellum (Baumann et al. 2007, Li et al. 2007,Shmerling et al. 1998). Overexpression of wild-type PrPC also caused disease in transgenic mice(Westaway et al. 1994).

Prion Disease in Humans and Animals

Prion diseases have occurred in humans and an-imals for many years. A disease similar to scrapiewas recorded in the mid eighteenth century,and scholars heavily debated its origin. A crucialexperiment showing incontrovertible trans-missibility of scrapie to goats was performed byCuille & Chelle` in the 1930s (Cuille & Chelle`1939). The rst cases of human prion disease,Creutzfeldt-Jakob disease (CJD), were reportedin the 1920s (Creutzfeldt 1920, Jakob 1921).The number of human and animal diseasesrecognized as TSEs has increased steadily andnow includes Gerstmann-Straussler-Scheinkersyndrome (GSS), fatal familial insomnia (FFI),

TSE: transmissiblespongiformencephalopathies

CJD: Creutzfeldt-Jakob disease

BSE: bovinespongiformencephalopathy

CWD: chronicwasting disease

FSE: felinespongiformencephalopathy

sCJD: sporadic CJD

and Kuru in humans; bovine spongiform en-cephalopathy (BSE) in cattle; chronic wastingdisease (CWD) in deer and elk; and trans-missible mink encephalopathy. BSE has beeninadvertently transmitted to a variety of captiveanimals, causing feline spongiform en-cephalopathy (FSE) and a plethora of diseasesin zoo animals including kudus, nyalas, andgreater cats, for example.

Creutzfeldt-Jakob disease. CJD was initiallydescribed as a sporadic disease occurring forno known cause (sCJD). The incidence ofCJD is low in all ethnicities and typically af-fects 1 person in one million each year. Veryrapid cognitive decline, causing dementia, is themain symptom. Cerebellar symptoms includ-ing ataxia and myoclonus are also frequent pre-senting symptoms. Death often occurs withinfew weeks of the rst signs of disease, anda fulminant, apoplectiform course of dis-ease has been documented in the past. So-matic mutations in the PRNP gene analogousto those in the germline of genetic CJD pa-tients (see below) have been hypothesized tounderlie sporadic CJD. Alternatively, Aguzzi& Glatzel (2006) suggested that some casesof alleged sCJD derive from heretofore un-recognized infections. Finally, PrPC may pos-sess a nite, albeit extremely low, propensity toself-assemble into ordered aggregates of PrPSc,thereby stochastically initiating prion replica-tion and, ultimately, a sporadic form of disease.The latter scenario could be regarded as thebad-luck hypothesis. However, none of this hasbeen proven, and therefore the cause of sCJDis still unknown.

Variant Creutzfeldt-Jakob disease andBovine Spongiform Encephalopathy. Pub-lic understanding of prion disease remainedlimited for a long time: For example, wehave heard neurologists saying that CJD isan essentially nonexistent disease. However,this mindset changed completely when BSEwas rst reported in the early 1980s (Wellset al. 1987). In the following years and untilmid 2007, BSE affected 190,000 cows

www.annualreviews.org The Prions Elusive Reason for Being 441

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vCJD: variant CJD

(http://www.oie.int/). Some investigatorssuggested that BSE could cause a new variantform of CJD (vCJD) in humans. A directexperimental proof that vCJD representstransmission of BSE prions to humans cannotbe produced. However, epidemiological,biochemical, neuropathological evidence andtransmission studies strongly suggest that BSEhas transmitted to humans in the form of vCJD(Aguzzi 1996, Aguzzi & Weissmann 1996,Bruce et al. 1997, Hill et al. 1997). The inci-dence of vCJD has been rising between 1994,when the rst patients suffering from vCJDpresented with their initial symptoms, and2001, raising fears that a very large epidemicmay be looming. At the time of this writing,vCJD has killed 200 individual victims world-wide (http://www.cjd.ed.ac.uk/). Most of theaffected individuals lived in United Kingdomand France. Fortunately, in the United King-dom the incidence appears to be decreasingfrom the year 2001 to 6 diagnosed cases yearlyin 2005 and 2006. In contrast, in France thenumber of probable and denite cases of vCJDincreased from 0 to 3 diagnosed cases per yearin 19962004 to 6 per year in 2005 and 2006. In2007, the number of cases was back to 3 again.(http://www.invs.sante.fr/publications/mcj/donnees mcj.html). A 30+-year mean in-cubation time of BSE/vCJD in humans isnot entirely implausible, and therefore someauthors have predicted a multiphasic humanBSE endemic with a second increase in the in-cidence of vCJD affecting people heterozygousat codon 129 (Collinge et al. 2006). Others,these authors included, regard the incidenceof vCJD as subsiding (Andrews et al. 2003)(Figure 1).

It is important to note, however, that theabove considerations apply primarily to the epi-demiology of primary transmission from cowsto humans. Although, by now a pool of pre-clinically infected humans may have been built.Human-to-human transmission may presentwith characteristics very different from thoseof primary cow-to-human transmission, in-cluding enhanced virulence, shortened incu-bation times, disrespect of allelic PRNP poly-

morphisms (129MM, MV or VV), and hetero-dox modes of infection including blood-bornetransmission. If we account for the time it willtake to eradicate these secondary transmissionsin the population, vCJD is not likely to disap-pear entirely in the coming four decades.

Iatrogenic CJD. Iatrogenic CJD is acciden-tally transmitted during the course of med-ical or surgical procedures. The rst docu-mented case of iatrogenic prion transmissionoccurred in 1974 and was caused by cornealtransplantation of a graft derived from a pa-tient suffering from sCJD (Duffy et al. 1974).Iatrogenic CJD is also rare, most often ob-served in individuals that have received cadav-eric dura mater implants and human growthhormone; some of these individuals receivedgonadotrophin extracted from human pitu-itary glands or had stereotactically placed elec-trodes in their brains (Will 2003). Four casesof vCJD transmission by blood transfusionshave been reported recently in the UnitedKingdom (Llewelyn et al. 2004, Peden et al.2004, Wroe et al. 2006) (see also http://www.cjd.ed.ac.uk/TMER/TMER.htm). The factthat preclinically infected individuals can trans-mit vCJD underscores the important medicalneed for sensitive diagnostic tools, which couldbe used for screening blood units prior to trans-fusion, for example.

Kuru. In the mid 1950s, when the remoteparts of Papua New Guinea were rst exploredby Australians and Westerners, Kuru was rstdescribed in research (Gajdusek & Zigas 1957).Kuru was, at that time and at least since 1941,an endemic disease among some tribes ofNew Guinea aborigines, especially among theFore linguistic group and neighboring tribes(Gajdusek & Reid 1961). Kuru in the Fore lan-guage means to shiver, and along with othersigns of cerebellar ataxia, shivering is a hallmarkof the disease. The ritual consumption of deadrelatives as a symbol of respect and mourningis the attributed route of transmission. As aconsequence, the incidence has steadily fallenafter cessation of cannibalism in Papua New


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0

5000

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gCJD: genetic CJD

OR: octarepeat region

Guinea (Collinge et al. 2006). In a concise andextremely clairvoyant observation published in1959, Bill Hadlow noted the epidemiological,clinical, and neuropathological similaritiesbetween Kuru and scrapie (Hadlow 1959).These were taken up by Carleton Gajdusekwho, in 1966, succeeded in transmitting Kuruto three chimpanzees (Gajdusek et al. 1966).Soon thereafter, serial passage of Kuru and ofseveral other prion diseases was demonstratedin chimpanzees and other primates (Gajduseket al. 1967, 1968). Investigators have sincetransmitted human prion disease to variousspecies including laboratory rodents.

Genetic CJD and Gerstmann-Straussler-Scheinker syndrome. Several mutations inthe prion protein gene (PRNP) have been

found in families with hereditary or geneticCJD (gCJD). Figure 2 summarizes known mu-tations causing human TSEs. gCJD occurswith point mutations mostly affecting the re-gion between the second and the third he-lix of the carboxy-terminus. However, inser-tions in the octarepeat region (OR) in theamino-terminus, and even one instance of apremature termination codon at position 145,have also been associated with human priondisease. The inheritance was, in all cases,autosomal dominant, often with very high pen-etrance. The clinicopathological disease phe-notype varies depending on the actual muta-tion, as well as on polymorphisms at codon 129,and most likely on a plethora of yet uniden-tied modiers and cofactors (Kovacs et al.2002).

CC1

CC2

HC H1 H2 H3 GPI OR

Mutations in PRNP associated with familial dementiaand/or neuropsychiatric symptoms(not further classified)

Mutations causing GSS Octarepeat insertion P102L-129M A117V-129V H187R-129V Q217R-129M

Mutations causing FFI D178N-129M

V180I E200K

T188K V210I

T188R-129V

E196K E211Q

V203I

Mutations causing gCJD Octarepeat insertion D178N-129V E208H M232R

P105L-129V G131V-129M F189S-129V

Y145*-129M D202N-129V

Q212P

G114V Q160*-129M N171S T183A

H187R

S S

Figure 2The human PrPC protein and its mutants. The mature human PrPC protein contains 208 amino acid residues. It features two positivelycharged amino acid clusters denoted CC1 and CC2 (blue boxes), an octapeptide repeat region (OR) ( green boxes), a hydrophobic core(HC) ( gray box), three -helixes (H1-H3) (red boxes), one disulphide bond (SS) between cysteine residues 179 and 214, and twopotential sites for N-linked glycosylation (red forks) at residues 181 and 197. A glycosylphosphatidylinositol anchor (GPI) ( yellow box) isattached to the C-terminus of PrP. This gure indicates in black framed boxes point mutations and insertions found in the humanPRNP gene in patients with prion disease. The associated polymorphisms of codon 129 (methionine M or valine V) are indicated.Amino acids are given in single-letter code. The asterisk indicates a stop codon; therefore, this mutation results in a truncated protein.


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The rst descriptions of Gerstmann-Straussler-Scheinker syndrome (GSS) origi-nate from 1928 and 1936 in an Austrian family(Gerstmann 1928, Gerstmann et al. 1936). Inthe following years, analogous disorders havebeen described, but its classication as a TSElagged until 1981, when Masters and colleagues(1981) reported that inoculation of brain tis-sue from three patients with GSS resulted inspongiform encephalopathy in nonhuman pri-mates. The authors also dened clinical hall-marks of GSS (earlier age at onset, longerdisease duration, and prominent cerebellarataxia) differentiating the disease from CJD.Nowadays GSS is considered an autosomal-dominantly inherited TSE caused by muta-tions in the prion protein open reading frame,manifesting typically with progressive cerebel-lar ataxia or spastic paraparesis and cognitivedecline. The known GSS-causing mutations aresummarized in Figure 2. In addition to the re-gions affected in gCJD, mutations altering thesequence of the central domain can cause GSS.Its distinctive neuropathological feature is thepresence of widespread large and multicentricamyloid plaques (Collins et al. 2001).

GSS is generally transmissible (Hsiao et al.1989, Masters et al. 1981, Tateishi et al. 1988);therefore, its classication as a TSE is widelyaccepted. However, the overall experimentaltransmissibility of GSS to nonhuman primatesand rodents is low. Only for the most com-mon GSS-associated mutations (P102L), andonly in approximately one third of the cases,were brain homogenates derived from patientsreproducibly capable of inducing disease upontransmission (Tateishi et al. 1996a). The lessfrequent mutations causing GSS often failedto induce disease after experimental transmis-sion to nonhuman primates and rodents, and inmany cases transmissibility was never assessed(Brown et al. 1994, Tateishi et al. 1996a).

Fatal familial insomnia. Fatal familial insom-nia (FFI) is the descriptive name given to a dis-ease identied in 1986. Five members of an Ital-ian family presented with insomnia and dysau-tonomia (Lugaresi et al. 1986). In 1992, the

GSS: Gerstmann-Straussler-Scheinkersyndrome

FFI: fatal familialinsomnia

disease-causing mutation in the prion proteingene (D178N) was identied, thereby allow-ing the classication of FFI as a genetically de-termined prion disease (Medori et al. 1992).The nal proof that FFI is a TSE was achievedwhen FFI was successfully transmitted to mice(Tateishi et al. 1995). FFI typically affects thethalamus, and accordingly, the core clinical fea-tures are disruption of the normal sleep-wakecycle, sympathetic overactivity, endocrine ab-normalities, and impaired attention (Collinset al. 2001). In addition to the pathogenic pointmutation D178N, the methionine-valine poly-morphism at codon 129 of the PRNP gene con-trols the disease phenotype. Whereas D178N-129MM (homozygosity for methionine atcodon 129) was associated with FFI, heterozy-gosity at codon 129 (D178N-129MV) segre-gated with the familial CJD subtype (Goldfarbet al. 1992). However, Zarranz et al. (2005)reported more recently that this genotype-phenotype association is not absolute. In onestudy, several patients have been identied witha CJD phenotype and a D178N-129MM geno-type. The authors concluded that rather thanbeing separate disease entities, prion diseasephenotypes such as FFI and CJD represent twoextreme manifestations of a continuous diseasespectrum (Zarranz et al. 2005).

In addition to the familial form of fatal in-somnia, a sporadic form of the disease, termedsporadic fatal insomnia, was described. Spo-radic FFI is not associated with mutations inthe PRNP gene (Mastrianni et al. 1999, Parchiet al. 1999).

The Nature of the Prion

Although formulated a century ago, Kochs pos-tulates remain the bedrock of microbiology. Ac-cording to Koch, three conditions must be metto identify a microbe as the causative agentof any given infection: (a) The microorganismmust be detectable in all diseased tissues, (b) itsisolation and growth must be achieved in pureculture, and (c) the culture-derived microorgan-isms must be able to induce disease after ex-perimental infection of a subject, from which


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PMCA: proteinmisfolding cyclicamplication

a further round of reisolation of the microor-ganism should be possible. Although Kochswork was performed long before contemporarymolecular biology, his postulates continue toserve remarkably well in dening conventionalviral and bacterial agents.

However, as prions are thought to be in-fectious proteins that amplify in a self-catalyticmisfolding process, their microbiological cul-ture sensu strictiori is not possible. Thereforewhether Kochs postulates can be meaningfullyapplied to prion disease is questionable. Fur-thermore, Kochs postulates account for the in-uence of host susceptibility, which is of utmostimportance in prion disease. Prion disease de-velopment depends on the presence of PrPC

on host cells, and the species-specic aminoacid sequence and polymorphism of codon 129are important. Alternate postulates for infec-tious proteinaceous agents have recently beensuggested (Walker et al. 2006), but it remainsto be seen whether they will garner universalacceptance.

In the prion eld, researchers generally ac-cept that a reasonable surrogate for Kochs sec-ond postulate be fullled by the generation ofsynthetic prions in vitro, i.e., the recovery ofperpetually transmissible infectivity from prionprotein produced recombinantly or chemicallyfrom dened constituents. Major progress to-ward this end has been made in recent days. Pu-ried PrPSc was used to generate PK-resistantPrP (PrPres) in a cell-free system that could evenreect two typical features of prions: speciesbarrier and strain specicity (Bessen et al. 1995,Kocisko et al. 1995).

Another approach used a method calledPMCA (protein misfolding cyclic amplica-tion), in which PrPres can be amplied byincubating and sonicating PrPres-containingbrain homogenate diluted in normal brain ho-mogenate. Soto and coworkers amplied PrPres

derived from scrapie-infected hamsters inde-nitely by using PMCA in serial dilutions. Am-plication of PrPres was accompanied by am-plication of infectivity (Castilla et al. 2005a).Certainly PMCA is a very sensitive method todetect PrPSc even in complex samples such as

blood and already in a presymptomatic diseasestate (Castilla et al. 2005b, Saa et al. 2006).The use of puried PrPC instead of brain ho-mogenate as a substrate decreased the efciencyof amplication, suggesting that additional co-factors may facilitate misfolding (Deleault et al.2005). For a long time, all attempts to use re-combinant PrP as a substrate for PMCA failed.However, Caughey and coworkers have nowsucceeded in carrying out PMCA using bacteri-ally expressed hamster PrP as a substrate. Whilethis represents a major advance in many ways,the sensitivity was not quite as high as that ofthe original PMCA (Aguzzi 2007, Atarashi et al.2007).

Infectivity may not have been generatedde novo in PMCA in these studies. Instead,prion-infected brain could have been inad-vertently added in the beginning. In an fas-cinating study, Supattapone and coworkersidentied the minimal components (PrPC, cop-uried lipids, and single-stranded polyanionicmolecules) required for amplication of PK-resistant PrP, and they convincingly showedthat prion infectivity can be generated de novoin brain homogenates derived from healthyhamsters using PMCA. Inoculation of furtherhealthy hamsters with the de novoformedprions caused a transmissible prion disease(Deleault et al. 2007). This study might be re-garded as the nal proof of the prion hypoth-esis. However, it also acknowledges PMCAslimitation for diagnostic purposes because PK-resistant material and infectivity can be formedin the absence of prions, thereby risking the re-porting of false positive results.

A second approach comprises de novogeneration of infectivity by misfolding re-combinant PrPC and subsequently inoculatingwild-type animals. In one attempt, a 55-residuepeptide encompassing the GSS mutationP101L was refolded in vitro to a beta-sheetrich peptide and could induce disease similarto GSS in transgenic mice expressing PrP(P101L). Transmission to wild-type mice wasnot successful, and PrP (P101L) was not resis-tant to PK. Because transgenic mice expressingPrP (P101L) develop disease spontaneously,


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although later in life than those exposed to thepeptide, Nazor et al. (2005) remarked that themisfolded peptide may have simply accelerateda spontaneously occurring disease.

Transmission to wild-type mice of an invitrogenerated misfolded part of the prionprotein (amino acid residues 89231) wasachieved a few years later. Legname andcoworkers produced PrP (89231) recombi-nantly and generated amyloid brils in vitro.These brils induced prion disease in trans-genic mice overexpressing PrP (89231), whichwas subsequently transmissible to wild-typemice (Legname et al. 2004, 2005).

Neurotoxicity

Current knowledge about the mechanisms be-hind neurodegeneration in prion disease andprionopathies is limited. Apoptosis and oxida-tive stress certainly contribute to some stages ofTSE pathology (Milhavet & Lehmann 2002),but little is known about damage causing pri-mary events. Early pathologic changes that oc-cur during prion disease involve synapses, yetthe molecular underpinnings of these ndingsremain unknown.

It is still unclear whether the toxicity ofPrPSc represents a gain of function or whetherloss of function of PrPC is responsible for neu-ropathological changes induced by prions. Al-though some authors belief that the toxicity inprion disease is explainable simply by a loss-of function of PrPC (Nazor et al. 2007), weand others (Westergard et al. 2007) believe again of function is more likely, particularly be-cause the phenotypes of PrPC-decient miceare very mild. However, a neuroprotective func-tion that may be physiologically provided byPrPC, which would protect neurons duringprion infection, could be reduced following itsconversion to PrPSc.

Our laboratory, and many others, has pur-sued the hypothesis that elucidating the phys-iological function of PrPC might help re-searchers understand the mechanism involvedin prion-induced neurodegeneration. The fol-lowing discussion centers on the discovery that

HC: hydrophobiccore

mice expressing deletion mutants of PrPC de-velop severe neurotoxic syndromes and identi-es the reasons why we believe that study ofthese syndromes may reveal the mechanismsoperative in prion diseases.

PHYSIOLOGICAL FUNCTION OFTHE CELLULAR PRION PROTEIN

The cellular prion protein PrPC is a GPI-linked extracellular membrane protein with twoN-linked complex glycosylation sites. PrPC ishighly abundant in the developing and ma-ture nervous system, where it is expressed byneuronal and glial cells. This mature versionoriginates from a precursor protein proteolyt-ically processed in the endoplasmic reticulumand Golgi (Stahl et al. 1987). As revealed by itsatomic structure, the mature PrPC protein con-tains a well-dened carboxy-terminal globulardomain comprising residues 127231 (murinenumbering), consisting of three alpha helicesand two beta sheets (Hornemann et al. 1997;Riek et al. 1996) and a structurally less-denedamino proximal region containing a stretch ofseveral octapeptide repeats, termed the OR,and framed by two positively charged chargeclusters, CC1 (aa 2327) and CC2 (aa 95110).These domains are linked by a hydrophobicstretch of amino acids [aa 111134, also termedhydrophobic core (HC)] (Figure 3).

Prion ProteinDeficient Mice

An astonishing number of independent linesof mice lacking PrPC have been generated byhomologous recombination in embryonic stemcells in many laboratories. Mice with disrup-tive modications restricted to the open readingframe are known as Prnpo/o [Zurich I] (Bueleret al. 1992) or Prnp/ [Edinburgh] (Mansonet al. 1994). They developed normally, and nosevere pathologies were observed later in life. Aspredicted by the protein-only hypothesis, thesemice were entirely resistant to prion infections(Bueler et al. 1993).

In contrast with these earliest lines, threelines generated afterwards: Prnp/ [Nagasaki],


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Rcm0, and Prnp/ [Zurich II] (Moore et al.1999, Rossi et al. 2001, Sakaguchi et al.1996) developed ataxia and Purkinje cell losslater in life. Because the phenotype wasabolished by reintroduction of Prnp as a

transgene, the originators of the Nagasakimice concluded that it occurred because ofthe lack of PrPC. This, however, wouldrun counter to the lack of pathology inPrnpo/o Zurich-I mice.

S S

S

CC1

SP CC2

HC H1 H2 H3 GPIORPheno-

type Trans-

mission Prion

propagation Rescue by PrP Refs.

0 23 32 80 90 107 121 134 177 200 217 231

PrPwt

no yes a PrP3280

no yes b;c PrP3293

no no b;d PrP32106

no yes* e PrP2388

no no e PrP238895107

no no e PrP2388108121

no yes e PrP2388141176

no no** f;g PrP114121

no yes h PrP104114

cerebellar disorder no no yes b PrP32121

cerebellar disorder no no yes b;d PrP32134

cerebellar disorder no nd yes f PrP94134

cerebellar disorder no nd yes k PrP105125

cerebellar disorder no yes no l PrP PG 14

nd e PrP 144 #

storage disease no no no e

PrP2388177200

storage disease no no no e

PrP2388201217

cerebellar disorder no no no e

PrP2388141221

nd e PrP2388122140

nd e PrP2388144 #

cerebellar disorder no no no j

PrP122231 #

no yes i PrP231 #

no no e PrP2388C178A A


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The discrepancy between the differentlines of PrP knockout mice was not resolveduntil a novel gene (Prnd ), encoding a proteincalled Doppel (Dpl), was discovered. Prnd islocalized 16 kb downstream of Prnp. In allthree lines of PrPC-decient mice developingataxia and Purkinje cell loss, a splice acceptorsite to the third exon of Prnp was deleted. Thisplaced Prnd under transcriptional control ofthe Prnp promoter, resulting in the formationof chimeric transcripts and in overexpressionof Dpl in the brain (Moore et al. 1999, Rossiet al. 2001, Sakaguchi et al. 1996). Preciselywhy the overexpression of Dpl is deleteriousis still unclear. On the basis of the observationthat Dpl expression induced heme oxygenase1 (HO-1) and neuronal and inducible nitricoxide synthases (nNOS and iNOS), suggestingan increased oxidative stress in the brainsof the Dpl-expressing Prnpo/o mice, Wonget al. (2001c) proposed that Dpl expressionexacerbates oxidative damage by antagonizingwild-type PrPCs antioxidative function.

The latency period before the various trans-genic mice overexpressing Dpl develop patho-logical phenotypes is inversely correlated to theDpl expression level in the brain, indicatinga rather strict gene-dosage effect (Rossi et al.2001). The Dpl-induced disease can be rescuedby coexpression of wild-type PrPC (Nishidaet al. 1999, Rossi et al. 2001), indicating thattoxicity of Dpl and the physiological functionof wild-type PrPC are not independent of eachother, but rather are involved in a commonpathway. Dpl-decient mice suffer from steril-

ity (Behrens et al. 2002), suggesting that the pri-mary physiological function of Dpl is related tosperm maturation.

Functional Domainsof the Prion Protein

For all the uncertainties surrounding the physi-ological and molecular functions of PrPC, someknowledge was generated by expressing a seriesof partially deleted Prnp variants in culturedcells and transgenic mice. Some of these mu-tants were made to identify the essential do-mains necessary for restoring prion susceptibil-ity. However, investigators found that domainexpression provoked spontaneous neurodegen-erative disease (Figure 3). In many instances,these syndromes were partially or fully coun-teracted by coexpression of wild-type PrPC. Be-cause the lack of PrPC itself did not induce anobvious phenotype, the latter pathologies indi-cate pathways in which PrPC is functionally ac-tive. Hence mice expressing PrP, which lacksdened domains, may allow for the identi-cation of functionally relevant domains withinPrPC.

N-terminal deletion mutants of PrP. TheOR has long been suspected to represent a ma-jor mediator of PrPCs function, and insertionmutations affecting the OR are associated withhereditary human prion disease. However,transgenic studies indicate that the OR isnot required for PrPC to function or for itsconvertibility into PrPSc (Flechsig et al. 2000).

Figure 3Murine PrPC protein and transgenic mutant PrP. Schematic drawing of full-length murine PrPC, includingthe signal peptide of the precursor protein (SP; brown box). Although amino acid numbering differs betweenhuman and mouse PrP, the organization of domains (including CC1 and CC2, OR, HC, and H1H3) issimilar to that of human PrPC (see Figure 2). Mouse PrP also contains a disulphide bond (SS) and aGPI-anchor. The left column denotes the individual mutants described in the text. The right columnsindicate presence or absence of phenotypic abnormalities (Phenotype) in transgenic mice when expressed ona PrP-decient genetic background, transmissibility of this phenotype to recipient mice (Transmission), andsusceptibility of transgenic mice to prions after intracerebral inoculation with a mouse-adapted strain ofscrapie prions. References: a, Fischer et al. (1996); b, Shmerling et al. (1998); c, Flechsig et al. (2000);d, E. Flechsig, I. Hegyi, A. Aguzzi, and C. Weissman (unpublished results); e, Muramoto et al. (1997);f, Baumann et al. (2007); g, Holscher et al. (1998); h, Hegde et al. (1998); i, Chesebro et al. (2005);j, Ma et al. (2002); k, Li et al. (2007); l, Chiesa et al. (1998).


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The OR appears to have, at best, a modulatinginuence on PrP conversion. Mice expressingOR-decient PrPC mutants do not developpathologies (Fischer et al. 1996, Muramotoet al. 1997, Shmerling et al. 1998). This wasunexpected because a variety of in vitro datahad identied the OR as being responsible forcopper binding (Aronoff-Spencer et al. 2000,Chattopadhyay et al. 2005, Furlan et al. 2007,Leclerc et al. 2006, Qin et al. 2002, Stockelet al. 1998; reviewed in Vassallo & Herms 2003)and for conferring protection against oxidativestress (Brown et al. 1999, Fukuuchi et al. 2006,White et al. 1999, Wong et al. 2001a). On theother hand, transgenic mice expressing ninesupernumerary octapeptide repeats, for a totalof 14 proline and glycine-rich repeats (Chiesaet al. 1998)which models a human familialCJD-linked mutationdevelop ataxia andcerebellar atrophy, granule cell loss, gliosis,progressive myopathy, and PrP deposition.The latter phenotype resembles its humancounterpart in some ways (Chiesa et al. 2000),yet transmission to wild-type mice failed(Chiesa et al. 2003).

In vitro studies indicate that the CC1 regionis involved in recycling and internalizing PrPC

from the cell surface (Sunyach et al. 2003,Taylor et al. 2005). Unfortunately, in vivolittle evidence supports the latter contention.Lack of CC1 in (PrP2388) (Muramoto et al.1997) did not induce pathologies in transgenicmice, and convertibility to PrPSc was retained.In PrP2388 mice, a second charge cluster(CC2) with several lysine residues aroundposition 100 may replace the function of CC1.However, mice bearing partial deletions ofCC2 (PrP2388 95107 and PrP2388 108121)are also healthy (Muramoto et al. 1997).The combination of amino-terminal deletionwith the elimination of amino acids 141176(PrP2388 141176) was also innocuous andrestored susceptibility to prion infection(Muramoto et al. 1996) despite a large deletionwithin the globular domain of PrPC.

The function of PrPC may depend on theHC region in concert with CC2. With the ex-ception of a small deletion between CC2 and

HC (PrP104114) (Hegde et al. 1998), abla-tion of CC2 in combination with a partial orcomplete deletion of HC elicits severe patholo-gies in mice. PrP32121 and PrP32134 trans-genic mice suffer from ataxia and cerebellargranule cell loss in addition to widespreadwhite matter disease (Radovanovic et al. 2005,Shmerling et al. 1998). The latter is also seenin mice expressing deletions encompassing all(PrP94134) or part (PrP105125) of the centraldomain (CD) (Baumann et al. 2007, Li et al.2007). These pathologies are radically differentfrom those seen in prion infections, and none ofthem goes along with pathological aggregationof PrP.

Each of these pathologies can be coun-teracted by coexpression of wild-type PrPC

(Baumann et al. 2007, Li et al. 2007, Shmer-ling et al. 1998), suggesting a competition ofsorts between PrPC and the toxic mutants. Inone conceivable scenario, PrPC and its variantsmay compete for a common ligand. Binding orcomplex assembly may represent the rst stepin a series of events that also involve the inter-action of an effector domain located in or con-trolled by the central domain (CC2 and HC),eventually resulting in signal transduction.

A partial deletion of HC (PrP114121)(Baumann et al. 2007) is nontoxic, but its po-tential to counteract the toxicity of PrP32134is lower than that of wild-type PrPC. Micewith deletion of CC2 and HC (PrP32121 andPrP32134 as well as PrP104114, PrP114121)did not support prion propagation (Flechsig &Weissmann 2004; Hegde et al. 1998, Holscheret al. 1998), indicating an involvement of theseregions in conversion.

Carboxy-proximal deletion mutants of PrP.Mice expressing PrP mutants with deletionsaffecting Helix 2 (PrP2388 177200), Helix3 (PrP2388 201217), or both helices 2 and3 (PrP2388 141221) suffer from ataxia andpresent with features of neuronal storage dis-ease (Muramoto et al. 1997, Supattapone et al.2001) but fail to replicate prions (Muramotoet al. 1996). Obviously at least Helix 2 and Helix3 are indispensable for stabilizing the structure


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of PrPC. None of these diseases proved to betransmissible to normal wild-type mice, andthey all manifested themselves independentlyof the presence or absence of wild-type PrP.This stands in sharp contrast to the group ofdeletion mutants affecting CC2 and HC.

Several attempts to generate mice express-ing truncated carboxy-terminal mutants lack-ing membrane anchoring (PrP2388 144# andPrP144#) have failed (Fischer et al. 1996,Muramoto et al. 1997). As in the case ofPrP2388 122140 (Muramoto et al. 1997), linesexpressing high levels of mRNA were gener-ated but protein was never detected. Essentialsignals may have been lost, thereby preventingcorrect sorting, processing, or folding of PrPand resulting in a short-lived polypeptide.

Mutations affecting the localization ofPrPC. The afnity of GPI-linked PrPC forarticial membranes, as measured by surfaceplasmon resonance (Elfrink et al. 2007), sug-gests extremely strong interactions. One mighttherefore expect that most PrPC is attached tocell membranes, with perhaps traces of PrPC

oating in body uids. Thus it may come asa surprise that plasma contains conspicuousamounts of PrPC (Volkel et al. 2001), and theconcentration of PrPC in cerebrospinal uidis even higher (Castagna et al. 2002). How-ever, it is unclear whether this soluble PrPC

is chemically identical to its membrane-boundisoform. Treatment of cultured cells with phos-phatidylinositol phospholipase C efciently re-leases PrPC from cultured cell membranes(Stahl et al. 1987), and a similar mechanism mayunderlie the physiological shedding of PrPC

into body uids.Release of full-length secreted PrPC was

forced by deletion of its carboxy terminal hy-drophobic domain (PrP231#), which is normallyreplaced by a GPI-anchor. This manipulationdid not induce any pathological phenotype(Chesebro et al. 2005). In contrast, targetingPrPC to the cytosol (cyPrP = PrP122 231#)by deleting its amino-terminal leader peptide(which targets PrPC to the endoplasmic retic-ulum and to the secretory pathway) provoked

ataxia with cerebellar degeneration and glio-sis (Ma et al. 2002). Coexpression of wild-typePrPC did not inuence the phenotypes of thesemice. Whether cytoplasmic expression of PrPand its cytotoxicity represent realistic modelsof the events occurring during prion disease re-mains very hotly debated (Fioriti et al. 2005,Roucou et al. 2003).

Point Mutations withinthe Prion Protein

As previously described, a considerable set ofpoint mutations within PRNP has been linkedto various forms of human prion diseases.Some of these mutations have been expressedin mice. With the possible exception of somestrains of mice expressing the P101L vari-ant of PrPC (Hsiao et al. 1994, Telling et al.1996), none of these attempts succeeded inreproducing the infectiousness of bona deprions. Point mutations affecting the two N-linked glycosylation sites of PrPC proved, as ex-pected, to alter its glycosylation (Kiachopouloset al. 2005). Point mutations N182T, A198T,or N182T/A198T prevented glycosylation intransgenic mice without grossly affecting cellu-lar sorting in cell culture. Mice developed nor-mally and were readily susceptible to scrapieor BSE (Neuendorf et al. 2004). Knock-in mu-tants carrying either N180T or N196T, orboth mutations, (Cancellotti et al. 2005) didnot suffer from any constitutive phenotype,even if the complete blockade of glycosylationby the N180T/N196T double mutation ledto a mainly intracellular localization of PrPC.This nding is somewhat surprising because re-sults from cultured cells had predicted that un-glycosylated PrPC would be prone to sponta-neous aggregation (Korth et al. 2000, Priola &Lawson 2001).

Two sets of point mutations, PrP3AV (ex-change of alanine to valine at positions 113,115, and 118) (Prusiner & Scott 1997) andPrPKHII (exchange of lysine 109 and histidine110 for isoleucine) (Hegde et al. 1998), gener-ated PrP with altered topology, termed CtmPrPin a cell free assay. CtmPrP supposedly spans


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the membrane via the HC domain (Hegdeet al. 1998). Transgenic mice expressing theseproteins developed a fatal neurological disor-der (Hegde et al. 1998). A similar phenotypewas observed in transgenic mice with substi-tution of leucine 9 into arginine in additionto this 3AV mutation (Stewart et al. 2005,Stewart & Harris 2005). However, researchnever formally proved that CtmPrP exists in vivo.It is still noteworthy that coexpression of wild-type PrPC with mutants promoting the CtmPrPtopology aggravated their phenotype. Subtlechanges, such as the removal of disulde bridges(PrP2388 C178A), are tolerated without induc-ing a spontaneous phenotype though reduc-ing the susceptibility for conversion into PrPSc

(Muramoto et al. 1997).

Evolution of the Prion Protein

PrP is present in a broad variety of species(Figure 4). Genes with similarities to Prnp existin birds (Gabriel et al. 1992), reptiles (Simonicet al. 2000), amphibians (Strumbo et al. 2001),and possibly in sh (Favre-Krey et al. 2007,Oidtmann et al. 2003, Rivera-Milla et al. 2003,Suzuki et al. 2002) in addition to all mammals.However, more primitive organisms such as in-sects, cephalopods, and protozoa have not beenreported to contain PrP homologs. All PrPsare glycosylated and membrane attached by aGPI anchor. The sequence identity among theknown PrP homologs is limited, and proteinlength can vary between 250 amino acids intetrapods to 600 amino acids in sh. Fishmay have developed additional Prnp-like genes(Rivera-Milla et al. 2006). The putative sh PrPgenes are thus far identied only on the basis ofrather tenuous sequence similarities. The con-tention that these molecules indeed representparalogs of PrPC would be greatly strengthenedif knockdown-induced phenotypes of zebrashwould be functionally corrected by mammalianPrPC expression. Such experiments have notbeen reported.

Comparisons between the available struc-tures and molecular models suggest that allPrPs share a common blueprint. A exible

amino-terminal tail, with a positively chargedCC1 at its far end and repetitive domainsof variable numbers, is hooked to a globu-lar carboxy-terminal domain. The fold of thisdomain is strongly conserved and stabilizedby a disulde bridge, although the primarysequence shows considerable diversity. Thesetwo domains are linked by a highly conservedhydrophobic linker having a second positive-charge cluster CC2 at its amino-terminus. Thislinker region is by far the most conserved se-quence motive of PrP in all species.

Cellular Processes Influencedby PrPC Expression

Several cellular processes in the nervous systemhave been inuenced by the Prnp-genotype, in-cluding neuronal survival; neurite outgrowth;synapse formation, maintenance, and func-tion; and maintenance of myelinated bers(Figure 5).

One of the most frequently suggested cel-lular functions of PrPC is a survival-promotingeffect on neuronal and nonneuronal cells, whichhas been observed in vitro as well as in in vivostudies.

This neuroprotective function, or cytopro-tective function in general reviewed in Roucou& LeBlanc (2005), has been mediated by anti-apoptotic or antioxidative mechanisms.

Antiapoptotic function. Neurons derivedfrom Prnp/ mice were originally reported tobe more susceptible to the induction of apop-tosis by serum-deprivation than were cells ex-pressing PrPC (Kuwahara et al. 1999), but thiseffect may have been brought about by Dploverexpression rather than by PrPC ablation.However, several studies indicate that PrPC

has a cytoprotective function by decreasingthe rate of apoptosis after particular apoptoticstimuli such as Bax overexpression or TNF-.Bax overexpression induces apoptosis in hu-man neuronal cells. Coexpression of wild-typePrPC, but not of PrP lacking the octarepeats,reversed the Bax-mediated induction of apop-tosis (Bounhar et al. 2001).


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The presence of PrP in the cytosol, be itdue to reverse translocation from the endoplas-mic reticulum or through direct cytosolic ex-pression, was virulently neurotoxic (Ma et al.2002). However, other studies failed to con-rm the toxicity of cytosolic PrP and claimedthat it can instead protect against Bax-mediatedapoptosis in human primary neurons (Roucouet al. 2003). In this context, PrPC inhibitedthe proapoptotic conformational change of Baxand cytochrome c release from mitochondria(Roucou et al. 2005).

In a screening approach for proteins pro-tecting cancer cells from apoptosis, researchersinvestigated the gene-expression prole in anestablished cell clone of MCF-7 breast cancercell line resistant to TNF-induced apoptosis.PrPC was overexpressed 17-fold. Conversely,overexpression of PrPC converted MCF-7 cellssensitive to TNF-induced apoptosis into re-sistant cells (Diarra-Mehrpour et al. 2004).

The neuroprotective function of PrPC inthe postischemic rodent brain has been inten-sively studied. Levels of PrPC after ischemiawere increased compared with controls (Shyuet al. 2005, Weise et al. 2004). Moreover,adenovirus-mediated overexpression of PrPC

reduced infarct size in rat brain and improvedneurological behavior after cerebral ischemia(Shyu et al. 2005). Conversely, in a mousemodel of ischemic brain injury Prnpo/o micedisplayed signicantly increased infarct vol-umes when compared with wild-type mice(McLennan et al. 2004, Weise et al. 2006). Twogroups of researchers showed that mice lackingPrPC had enhanced postischemic caspase-3 ac-tivation (Spudich et al. 2005, Weise et al. 2006).An increase in Erk-1/-2, STAT-1, and JNK-1/-2 phosphorylation and activation was iden-tied, suggesting PrPCs possible involvementin cellular signaling (Spudich et al. 2005). Also,a reduced amount of phospho-Akt in the graymatter suggested that PrPC deciency bringsabout an impairment of the antiapoptotic phos-phatidylinositol 3-kinase/Akt pathway (Weiseet al. 2006). Finally, Mitteregger et al. (2007)claimed that the OR is required within PrPC

for the neuroprotection in the ischemic mouse

brain, although the genetic homogeneity of themice tested in the latter experiment was notcontrolled for.

Protection against oxidative stress. Besidesits possible antiapoptotic function, there aremany reports about an antioxidative effect ofPrPC. These two effects are not necessarily mu-tually exclusive. Oxidative stress may be in-volved in TSE pathogenesis. However, onemust remember that oxidative stress is very un-specic and is seen in different kinds of damageto the nervous system with impaired mitochon-drial function such as defects in the ubiquitin-proteasome system, protein aggregation, andinammation.

Many investigators believe that the mainfunction of PrPC consists of protecting againstoxidative stress (see Milhavet & Lehmann 2002for a review). First hints came from in vitrostudies of rat pheochromocytoma cells. Thoseselected for resistance to copper toxicity oroxidative stress showed higher levels of PrPC

(Brown et al. 1997a). Primary neuronal cellslacking PrPC were more susceptible to hydro-gen peroxide (H2O2) than were wild-type cells.The increased peroxide toxicity went along witha signicant decrease in glutathione reductaseactivity measured in PrPC-decient neurons(White et al. 1999). Also, PrPC-decient pri-mary neurons were more susceptible to treat-ment with agents inducing oxidative stress com-pared with wild-type cells, a phenomenon thatwas explained by a reduced Cu/Zn superox-ide dismutase (SOD) activity observed in vivo(Brown et al. 1997b, 2002). Higher levels of ox-idative damage to proteins and lipids were iden-tied in the brain lysates derived from Prnp/

compared with wild-type mice (Klamt et al.2001, Wong et al. 2001b).

PrPC itself could have SOD activity andthereby mediate the antioxidative function(Brown et al. 1999). However, there is sig-nicant controversy about this alleged SODactivity. Others, ourselves included, failed toconrm this proposed SOD activity in vitro(Jones et al. 2005) and in vivo (Hutter et al.2003). Furthermore, PrPC expression level did


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4.01 165 330 494 658

3.2

2.4

1.6

0.8

0

0.8

1.6

2.4

3.2

4.0 DeerSheepCowCamelRabbitBatChimpanzeeHumanMouseSigmodonPigeonQuailChickenDuck

Zebrafish_prp1Trout_prp1Carp_prp1Zebrafish_prp2Frog

Position

Multiple alignment

a

b

Tetrapod PrP Fish PrP

Hum. Prp253 aa

S R H T N N

S--S

Fug. Prp1461 aa

S R H T N

S--S

S R T N N Zeb. Prp1606 aa

S--S

H

S R H T N N

S--S

Chi. Prp273 aa

S R H T N N

S--S

Xen. Prp216 aa

S R H T N N

S--S

Tur. Prp270 aa

Sal. Prp605 aa

S R

S--S

H N T

Zeb Prp2567 aa

S R

S--S

H N N T

Fug. Prp2435 aa

Hyd

rop

ho

bic

ity

S R

S--S

H H N T

Turtle

Figure 4PrP structural diversity in vertebrates. (a) Schematic drawing of tetrapod PrPs and long (PrP1 and PrP2) sh PrPs. The speciesabbreviations refer to sequences from human (Hum), chicken (Chi), turtle (Tur), Xenopus (Xen), zebrash (Zeb), salmon (Sal), andFugu (Fug). The location and relative size of conserved structural features are indicated. However, these features were physicallydetermined for the structure of human PrPC and represent mere conjectures in the case of sh. Domains are indicated by differentboxes and/or letters: S, signal peptide sequence; R, repetitive region; H, hydrophobic region; SS, disulde bridge; N, glycosylationsite; arrow, GPI anchor residue; and T, hydrophobic tail. (b) Comparison of hydrophobicity plots. Sequences of indicated species werealigned using DNAMAN software (Lynnon BioSoft, Canada), and a hydrophobicity plot was generated using a window of nineamino-acid residues. Numbering of residues is according to alignment matrix. (c) 3-dimensional structures of human (hum based on1QM2.pdb model) chicken (chi based on 1U3M.pdb) turtle (tur based on 1U5L.pdb), and frog (fro based on 1XOU.pdb); pdb les arefrom the protein database (Berman et al. 2000). Note the similarity of the carboxy-terminal globular domain. (d ) Evolutionaryrelationships among vertebrate PrP sequences are based on distance methods (neighbor-joining). Bootstrap values are shown at relevantnodes using DNAMAN software (Lynnon BioSoft, Canada).


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not signicantly inuence SOD activity invivo (Hutter et al. 2003, Waggoner et al.2000).

Mitochondria play an important role notonly in oxidative stress but also in the inductionof apoptosis. Morphological alterations in mi-tochondria have been described in scrapie-infected hamsters (Choi et al. 1998) and mice(Lee et al. 1999) as well as in mice lacking PrP,in which the number of mitochondria was re-duced (Miele et al. 2002).

Role of PrPC in synapses. Synapses have de-veloped into a sort of hot spot in prion research.Several immuno-electron microscopy studiescould show that PrPC is localized in synap-tic boutons, whereas it is mainly presynaptic(Fournier et al. 1995, Moya et al. 2000, Saleset al. 1998, Tateishi et al. 1996b). However,others described a much broader distribution ofneuronal PrPC (Laine et al. 2001, Mironov et al.2003). Because PrPC is processed and brokendown into various fragments, not all of which

PrPhum PrPchi PrPtur PrPfro

PigeonQuail

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Figure 4(Continued )


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Neuronal survival Protection against apoptosis Protection against oxidative stress

Neurite outgrowth Dendrites Axons

Synapse Formation Function

Maintenance ofmyelinated axons

Figure 5Physiological processes involving PrPC. Several processes in the nervous system have been inuenced by PrPC. Neurite outgrowth,including growth of axons and dendrites, was observed to be reduced in neurons lacking PrPC. PrPC has often been reported topromote neuronal survival, in particular following apoptotic or oxidative stress. Cerebellar granule cell apoptosis was observed in miceexpressing toxic N-terminal deletion mutants of PrP. In addition, the latter transgenic mice show an impaired maintenance ofmyelinated axons in the white matter. Another site of PrPC action might be the synapse, which is often affected in the rst stage ofprion diseases and whose formation was found to be reduced in neuronal cultures devoid of PrPC. Furthermore, electrophysiologicalstudies indicate a role of PrPC in synapse function, especially in neurotransmitter release.

are recognized by the antibodies used in thesestudies, one might speculate that some PrPC

degradation products acquire distinct subcellu-lar topologies.

Early pathologic changes occurring in priondiseases involve synapse loss and PrPSc depo-sition in synaptic terminals (Grigoriev et al.1999, Jeffrey et al. 2000, Kitamoto et al. 1992,Matsuda et al. 1999, Roikhel et al. 1983). Synap-tic vesicle proteins associated with exosomesand neurotransmission are reduced in brainsof patients with spongiform encephalopathy(Ferrer et al. 1999). Synaptic disorganizationand loss are fundamental and constant fea-tures of prion disease, irrespective of the pres-ence or absence of spongiform change, neu-ronal loss, and severe gliosis (Clinton et al.1993). Abnormal electrophysiological record-ings in scrapie-infected mouse and hamster hip-pocampal and cortical slices further supportthe synaptic dysfunction during the course ofprion disease (Barrow et al. 1999, Johnston et al.1998). In a terminal disease state, PrPSc accu-mulation in synaptosomes correlated with alter-ations in the GABAergic system (Bouzamondo-Bernstein et al. 2004). Despite the wealth of

the above evidence, however, it should not goundiscussed that synaptic changes can representnonspecic phenomena that are seen in essen-tially all brain diseases at one stage or another.

The generally held view that PrPC is animportant protein in synapses is supportedby electrophysiological studies of CA1 hip-pocampal neurons derived from PrPC-decientmice. Excitatory glutamatergic synaptic trans-mission, GABAA receptormediated fast inhibi-tion, long-term potentiation, and late afterhy-perpolarization were reduced or absent in micelacking PrPC (Carleton et al. 2001; Collinget al. 1994, 1996; Mallucci et al. 2002). Some ofthe ndings could be explained by alterationsin Ca-activated K+ currents (Colling et al.1996, Herms et al. 2001). However, the readershould note that alterations in synaptic trans-mission were not conrmed by others (Lledoet al. 1996), and glutamatergic synaptic trans-mission was even observed to be increased inPrPC-decient mice by yet another laboratory(Maglio et al. 2004, 2006). Another report in-dicates the impact of aging on these alterationsdescribing a reduction in the level of postte-tanic potentiation and long-term potentiation


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only in old PrPC-decient mice (Curtis et al.2003). In summary, the impact of the loss ofPrPC on hippocampal electrophysiological pa-rameters is still being hotly debated despite afull decade of research efforts. Some of the dis-crepancies may depend on additional geneticmodiers for which investigators have not rig-orously controlled.

Other alterations in PrPC-decient micemight be related to synaptic dysfunctionsuch as altered circadian rhythms and sleep(Tobler et al. 1996) and impaired hippocampal-dependent spatial learning (Criado et al. 2005).The neuromuscular junction is another sitewhere PrPC was concentrated, namely en-riched in subsynaptic endosomes (Gohel et al.1999). A potentiation of acetylcholine releasefrom presynaptic axon terminals was observedafter administration of recombinant PrP atnanomolar concentrations to mouse phrenic-diaphragm preparations (Re et al. 2006). Thesuggestion of an involvement of PrPC insynapse formation originated from in vitro ob-servations in hippocampal neurons, in whichsynaptic-like contacts were increased after addi-tion of recombinant PrP (Kanaani et al. 2005).

It is unknown whether the role of PrPC insynapses is related to its above-mentioned an-tiapoptotic or antioxidative effects or whetherit is mainly the involvement of PrPC in neu-rotransmitter release (e.g., via direct interac-tion with synapsin1 and synaptophysin). How-ever, an as-yet-unidentied process could alsoplay a key role, or several processes could worktogether.

Neurite outgrowth. Several lines of evidenceindicate PrPCs involvement in neuronal de-velopment, differentiation, and neurite out-growth. Axon or dendrite outgrowth was asso-ciated with PrPC-dependent activation of sig-nal transduction pathways including p59Fyn ki-nase, cAMP/protein kinase A (PKA), proteinkinase C (PKC), and MAP kinase activation(Chen et al. 2003, Kanaani et al. 2005, Lopeset al. 2005, Santuccione et al. 2005). P59Fynkinase activation in this context was depen-

dent on the recruitment of neural cell adhesionmolecule (N-CAM) to lipid rafts (Santuccioneet al. 2005). Recent studies show that PrPC pos-itively regulates neural precursor proliferationduring development and adult mammalian neu-rogenesis (Steele et al. 2006).

Maintenance of the white matter. Centralnervous system white matter, composed mainlyof myelinated axons, might be disrupted inprion diseases and prionopathies. In some casesof GSS, cerebellar and frontal white matter areaffected (Itoh et al. 1994). In an experimentalmodel of human TSEs in rodents, vacuolationin myelinated bers with splitting of myelinlamellae was observed (Walis et al. 2003). PrPC

is present in puried myelin fractions derivedfrom brain homogenates (Radovanovic et al.2005). Several transgenic mice expressing dele-tion mutants of PrPC (Baumann et al. 2007, Liet al. 2007, Shmerling et al. 1998) as well asPrnp/ mice accidentally overexpressing Dpl(Nishida et al. 1999) show vacuolation and de-generation of myelinated bers in the centralnervous system.

PrP and the Immune System

The immune system plays a fundamental role inprion disease and PrPC is expressed on cells ofthe immune and hematopoietic system, where itmight have a physiological function. This topicis reviewed in depth in Isaacs et al. (2006). Also,Zhang et al. (2006) reported that PrPC is in-volved in self-renewal of hematopoietic stemcells.

Molecular Mechanisms Mediatingthe Function of PrPC

Despite the overwhelming number of re-ports about alterations in mice and cells lack-ing PrPC summarized above, little is knownabout the molecular mechanisms involved inthese cellular processes. Figure 6 depicts sometheoretical models of how PrPC might inu-ence cell signaling, endocytosis, and cell ad-hesion. Whether these events are mutually


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exclusive, or whether they occur only underspecic circumstances in a diversity of tissues,or whether they can act in a combined way,remains speculative. In all cases, PrPC is likely

to mediate its function via one or more interac-tion partners.

One explanation for the diversity of the sug-gested physiological functions of this PrPC is

Interaction with TM protein in trans

Modulation of signaling pathways

Modulation of cellular adhesion

Endocytosis of PrPc via Clathrin-coated pits or caveolae

Cointernalization of anothercell component

Modulation of signaling pathways

Degradation of PrPc

cointernalized TM proteins

Interaction with TM protein in cis

c

a

b

Modulation of signal transduction pathways, e.g.activation of:

Fyn kinase

Erk1/2

cAMP

PKC

Lipid raft Non-raft region

Lipid raft Non-raft region


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that it may exert pleiotropic effects, therebymodulating the function of several cellularpathways. Examples for such a general cellu-lar process would be stabilization of proteincomplexes and the targeting of cell compo-nents to certain cellular sites, such as rafts orendosomes.

Signaling. The attachment of PrPC to themembrane by a GPI anchor, its localization indetergent-resistant membranes, also known aslipid rafts, in many cell types may suggest an in-volvement in cellular signaling (Shmerling et al.1998) as is the case for other raft-associatedproteins. Moreover, as we describe below, PrPC

could also inuence cellular signaling events byits involvement in endocytotic pathways.

Several signaling pathways or signalingcomponents, such as Akt, Fyn, cAMP, andErk1/2, are modulated by PrPC expression, itscross-linking, or its interaction with anotherprotein. Antibody-mediated cross-linking ofPrPC induced activation of the p59Fyn ki-nase, a family member of nonreceptor Src-related kinases, in neuronal differentiated cellsin a caveolin-1-dependent manner (Mouillet-Richard et al. 2000). As a downstream event,the same group claims to have identied Erk1/2phosphorylation (Schneider et al. 2003). PrPC

cross-linking additionally modulates seroton-ergic receptor activity in differentiated neu-ronal cells (Mouillet-Richard et al. 2005). Thending that PrPC cross-linking modulates ac-tivity of serotonergic receptors in differenti-ated neuronal cells await replication and invivo conrmation. However, p59Fyn activationand downstream activation of Erk1/2 were also

seen in a hypothalamic cell line (Toni et al.2006).

Several studies indicated PrPC involvementin neurite outgrowth and neuronal survival.Chen et al. reported increased neuronal survivaland neurite outgrowth from neurons when cul-tured on Chinese hamster ovary (CHO) cellstransfected to express mouse PrP. Althoughp59Fyn kinase activity in this context was in-volved mainly in neurite outgrowth, the PI3 ki-nase/Akt pathway as well as regulation of Bcl-2and Bax expression contributed to the survivaleffect elicited by PrP. Cyclic AMP/protein ki-nase A (PKA) and Erk signaling pathways con-tributed to both neurite outgrowth and neu-ronal survival (Chen et al. 2003, Santuccioneet al. 2005).

Some investigators suggested that engage-ment of PrPC with stress-inducible proteinI (STI1) induces neuroprotective signals thatrescue cells from apoptosis via cAMP/proteinkinase A and the Erk signaling pathways(Chiarini et al. 2002, Zanata et al. 2002). In-teraction with STI1 induced different signalingpathways, promoting neuroprotection by PKAactivation and neuritogenesis by activation ofthe MAPK pathway (Lopes et al. 2005).

Endocytosis and internalization of PrPC.PrPC is rapidly internalized from the cellmembrane. This internalization of PrPC couldbe crucial for its function, e.g., by inuenc-ing signal transduction pathways. Endocyto-sis of membrane receptors does not neces-sarily downregulate receptor activity. Whilebeing internalized, both tyrosine kinase andG proteincoupled receptors may remain

Figure 6Models of how PrPC could exert its physiological function. (a) Endocytosis of PrPC via clathrin-coated pits or caveolae may represent a mechanism for the downregulation of PrPC on the cell surface.Alternatively, or additionally, endocytosis of PrPC leads to cointernalization of another cell component, e.g.,a proteinaceous interacting partner, thereby regulating the presence of the latter on the cell surface. Thisregulation could positively or negatively modulate the activity of signal transduction pathways, e.g., viainducing degradation of the cointernalized partner. (b) An interaction with a transmembrane (TM) protein incis independent of an internalization process may lead to modulation of signal transduction pathways in thecell carrying PrPC on its cell surface. (c) Similarly, an interaction with another protein in trans may lead tomodulation of signal-transduction pathways or adhesion to adjacent cells.


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activated and produce intracellular responsesalong the endosome-lysosome pathway (Pradoet al. 2004). Internalization of tyrosine kinasereceptors was functionally important in stud-ies: TrkA receptors, for example, mediate nervegrowth factor (NGF)-dependent cell survivalwhile they are located at the cell membrane,whereas internalization is required for induc-tion of neurite outgrowth (York et al. 2000,Zhang et al. 2000).

The mechanism of PrPC internalization isstill controversial because both raft/caveolae orcaveolae-like (Kaneko et al. 1997, Marella et al.2002, Peters et al. 2003, Vey et al. 1996) as wellas clathrin-dependent endocytosis may be op-erative (Shyng et al. 1994, Taylor et al. 2005).These mechanisms might as well be equallyimportant. In addition, internalization of thesame ligand/receptor complex by distinct endo-cytotic pathways can result in different signal-ing outcomes. TGF- receptor, for example, isdegraded after endocytosis via caveolae, but in-ternalization via clathrin-coated pits promotesits signaling (Di Guglielmo et al. 2003). How-ever, in lymphocytes and neuronal cells that donot express caveolin, internalization can occurin a lipid raftassociated noncaveolar, clathrin-independent process (Kirkham & Parton 2005,Parton & Richards 2003). Therefore addi-tional, less well-characterized endocytosis path-ways including caveolae-like endocytosis mightbe involved in internalization of PrPC.

For endocytosis by clathrin-coated pits,PrPC would need to leave the lipid rafts priorto internalization because the rigid structure ofraft lipids is unlikely to accommodate the tightcurvature of coated pits. This phenomenonoccurred after binding of copper to the OR(Sunyach et al. 2003, Taylor et al. 2005), butits physiological signicance is unknown. Low-density lipoprotein receptor-related protein 1(LRP1) was later shown to mediate PrPC en-docytosis (Taylor & Hooper 2007), and CC1region was essential for its internalization inneuroblastoma cells (Sunyach et al. 2003, Tay-lor et al. 2005). Sunyach et al. (2003) suggestedthat heparin sulfate proteoglycans are partof the endocytotic complex involving PrPC.

Glypican-1, a GPI-anchored heparan sulfate-containing cell-associated proteoglycan, inter-acts and cointernalizes with PrPC in N2a afterinduction with copper ions. In cells express-ing PrP, which lacks the OR, internalization ofglypican-1 is reduced, suggesting a possible rolefor PrPC in the cointernalization of other cel-lular components (Cheng et al. 2006).

PrPC might participate in the correct local-ization of some other proteins in lipid rafts.Neuronal nitric oxide synthase (nNOS), for ex-ample, involved in various nervous system pro-cesses such as development, synaptic plasticity,regeneration, and regulation of transmitter re-lease was associated with lipid rafts in wild-typemice. In contrast, in brains of PrPC-decient aswell as scrapie-infected mice, nNOS was not as-sociated with rafts, and activity of nNOS was re-duced. Therefore PrPC could be important forthe proper cellular localization of other proteins(Keshet et al. 1999). Similarly N-CAM was re-cruited to lipid rafts by PrPC (Santuccione et al.2005).

However, PrPC is involved in a number ofcellular functions and how endocytosis inu-ences them in vivo remains widely unknown; in-ternalization of PrPC could contribute to down-regulation of a signaling event but could also benecessary for signaling. A general involvementof PrPC in vesicle formation could be a possibleexplanation for most of the suggested molecu-lar functions of PrPC because it could regulatesignaling and inuence synaptic transmission.

PrP and cell adhesion. Several reports areconsistent with a possible function of PrPC

as a cell adhesion or recognition molecule.Some interaction partners of PrPC identiedso far have a role in adhesion, including laminin(Graner et al. 2000a,b), a structural componentof basement membrane, laminin-receptor pre-cursor (Gauczynski et al. 2001, Rieger et al.1997), and N-CAM (Schmitt-Ulms et al. 2001).These three molecules are involved in adhe-sion in a diversity of signal transduction path-ways, in differentiation, and in neurite out-growth (Colognato & Yurchenco 2000, Maness& Schachner 2007). Interaction of laminin


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or N-CAM with PrPC has been associatedmainly with its suggested role in neuritogenesisand neurite outgrowth (Graner et al. 2000a,b;Santuccione et al. 2005). Sales et al. (1998)also proposed that PrPC in its synaptic locationmight stabilize apposing synaptic membranesthrough adhesive mechanisms.

The evidence reported so far could indicatethat PrPC is involved in adhesive mechanisms,but this is likely not its sole function. Adhe-sion molecules that interact biochemically withPrPC could also transduce a PrPC-dependentsignal. The neurite-outgrowth-promoting in-teraction between PrPC and N-CAM, for ex-ample, was associated with activation of thep59Fyn kinase (Santuccione et al. 2005).

Interaction Partners of PrPC

Investigators often hypothesized that PrPC ex-erts its function via interaction with othercell-surface components. GPI-anchored pro-teins would need to interact with a transmem-brane adaptor to inuence intracellular signal-transduction pathways, thereby enabling thetransduction of an extracellular signal. An ex-ample of such a protein is the urokinase-typeplasminogen activator receptor uPAR, whichis involved in cellular adhesion, differentia-tion, proliferation, and migration mediated bythe interaction with transmembrane adaptorssuch as integrins, G proteincoupled receptors,and caveolins (Blasi & Carmeliet 2002). Analo-gously, PrPC may bind to a transmembrane pro-tein or to a protein complex that mediates func-tional association with intracellular pathways.

The toxic deletion mutants of PrPC maydestroy such a complex by competing for thebinding of some complex components yet fail-ing to interact with the signal-transducing com-ponents. Indeed, several models for the toxi-city of PrPC deletion mutants have proposedthat PrPC binds to a transmembrane receptorand that deletion mutants induce a toxic sig-nal (Li et al. 2007) or prevent a survival signal(Baumann et al. 2007, Shmerling et al. 1998).

The commonly shared opinion that PrPC

binds to a receptor might explain the tremen-

dous effort put into the identication of its in-teraction partners by different methods such asyeast-two hybrid, coimmunoprecipitations, andcross-linking experiments.

All PrPC interaction partners identied thusfar are summarized in Table 1. They in-clude membrane proteins (receptors, enzymes,Caveolin-1, Na-K-ATPase, and a potassiumchannel), cytoplasmic proteins (componentsof the cytoskeleton, heat-shock proteins, andadaptor proteins involved in signaling), andeven the nuclear protein CBP70. Tantalizingly,several of these interactions partners are knownto play a role in synaptic vesicle function. Un-fortunately, the physiological relevance of mostof the proposed interaction partners remainsunconrmed. Proteins that are not even lo-calized to the outer leaet of the cell mem-brane, where PrPC is located and believed toexert its function, would at least require an addi-tional interacting cell component, meaning thattheir interaction with PrPC must be indirectat best.

Another possible explanation for cytosolicinteraction partners is the suggested presenceof transmembranous variants of PrPC, termedNtmPrP and CtmPrP. However, under normalconditions they have been described at best inminute amounts only (Hegde et al. 1998, 1999;Stewart & Harris 2001). Cytosolic PrP was laterdetected (Ma & Lindquist 2002, Ma et al. 2002).The signicance of these ndings still remainsobscure.

One must consider methodical bottlenecks.Because PrPC is exposed to the extracellularspace, it is questionable whether a yeast two-hybrid screen that articially exposes PrPC tothe cytosolic compartment with its differentbiochemical composition is the most appropri-ate method by which to study PrPC interactionpartners. A high number of false-positiveresults is to be expected, and therefore it isparticularly important that additional methodsare used to conrm interactions identied byyeast-two hybrid.

One item of paramount importance in im-munoprecipitation experiments is the choiceof the detergent conditions to allow for weak


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Table 1 Molecules identified as interaction partners to PrPC or PrPSc a

Interactionpartner Subcellular localization Method Binding site

Function of interaction partner andreferences

Hsp60 and GoEL Mitochondria Y2H 180210 Chaperone (Edenhofer et al. 1996)Laminin receptorprecursor

Plasma membrane Y2H, Co-IP 144179 Cell adhesion (Hundt et al. 2001,Rieger et al. 1997)

Laminin Extracellular, basementmembrane

Binding assay Unknown Signal transduction, cell adhesion,neuritogenesis (Graner et al. 2000a,b)

Synapsin Ib Synaptic vesicles Y2H, Co-IP 23100 and90231

Synaptic vesicle formation, modulation ofneurotransmitter release and in synap-togenesis (Spielhaupter & Schatzl 2001)

Grb2 Cytoplasm Y2H, Co-IP 23100,90231

Adaptor protein mediating signal transduc-tion (Spielhaupter & Schatzl 2001)

Pint1 Unknown Y2H, Co-IP 90231 Unknown (Spielhaupter & Schatzl 2001)N-CAM Plasma membrane

(transmembrane/GPI-anchored)

Formaldehydecross-linking

141176 Cell adhesion, signaling, etc. (Maness &Schachner 2007, Schmitt-Ulms et al.2001)

Stress-inducibleprotein 1 (STI 1)

Cytoplasm, plasmamembrane?

ComplementaryhydropathyCo-IP

113128 Heat shock protein (Chiarini et al. 2002,Zanata et al. 2002)

Caveolin-1 Caveolae, plasma membrane(transmembrane)

Co-IP unknown Caveolae formation and endocytosis;cross-linking of PrP induces Fynactivation (Mouillet-Richard et al. 2000)

Fyn kinase Cytoplasm, associated withcytosolic side of plasmamembrane

Co-IP unknown Signal transducer molecule (Mattei et al.2004, Mouillet-Richard et al. 2000)

ZAP70 Cytoplasm Co-IP Unknown Signal transduction during T cellactivation (Mattei et al. 2004)

Synaptophysin Synaptic vesicles(transmembrane)

Co-IP Unknown Presynaptic vesicle protein (Keshet et al.2000)

Neuronal nitricoxide synthase(nNOS)

Intracellular, partlymembrane bound

Co-IP Unknown Production of NO in neuronal tissue,involved in signaling, neurotransmission,etc. (Keshet et al. 2000)

-dystroglycan Plasma membrane(transmembrane, part of thedystroglycan complex)

Co-IP Unknown Transmembrane protein, bindsextracellularly to -dystroglycan (boundto laminin) and, intracellularly, todystrophin (Keshet et al. 2000)

Dp71 (dystrophin) Cytoplasmic face of plasmamembrane (part of thedystroglycan complex)

Co-IP Unknown Cytoskeletal protein (Keshet et al. 2000)

-syntrophin Cytoplasmic

the prion's elusive reason for being

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