Highly neurotoxic monomeric α-helical prion proteinMinghai Zhou, Gregory Ottenberg1, Gian Franco Sferrazza1, and Corinne Ida Lasmézas2

Department of Infectology, The Scripps Research Institute, Jupiter, FL 33458

Edited* by Charles Weissmann, The Scripps Research Institute, Jupiter, FL, and approved January 13, 2012 (received for review November 8, 2011)

Prion diseases are infectious and belong to the group of proteinmisfolding neurodegenerative diseases. In these diseases, neuro-nal dysfunction and death are caused by the neuronal toxicity ofa particular misfolded form of their cognate protein. The ability tospecifically target the toxic protein conformer or the neuronaldeath pathway would provide powerful therapeutic approachesto these diseases. The neurotoxic forms of the prion protein (PrP)have yet to be defined but there is evidence suggesting that atleast some of them differ from infectious PrP (PrPSc). Herein, with-out making an assumption about size or conformation, wesearched for toxic forms of recombinant PrP after dilution refold-ing, size fractionation, and systematic biological testing of all frac-tions. We found that the PrP species most neurotoxic in vitro andin vivo (toxic PrP, TPrP) is a monomeric, highly α-helical form ofPrP. TPrP caused autophagy, apoptosis, and a molecular signatureremarkably similar to that observed in the brains of prion-infectedanimals. Interestingly, highly α-helical intermediates have beendescribed for other amyloidogenic proteins but their biologicalsignificance remains to be established. We provide unique exper-imental evidence that a monomeric α-helical form of an amyloido-genic protein represents a cytotoxic species. Although toxic PrPhas yet to be purified from prion-infected brains, TPrP might bethe equivalent of one highly neurotoxic PrP species generatedduring prion replication. Because TPrP is a misfolded, highly neu-rotoxic form of PrP reproducing several features of prion-inducedneuronal death, it constitutes a useful model to study PrP-inducedneurodegenerative mechanisms.

misfolded protein diseases | neurotoxicity | neurodegeneration

Transmissible spongiform encephalopathies or prion diseasesare fatal brain diseases of animals and humans and belong to

the group of protein misfolding neurodegenerative diseases. Notreatment is available to halt the rapidly progressing neurode-generative disease once prions have reached the brain. PrPSc, anabnormally structured and aggregated form of the host prionprotein PrP (1, 2), is thought to be the only (3–6) or the major (7)constituent of the infectious agent called prion. PrPSc consists ofa proteinase K (PK)-resistant and a PK-sensitive form (rPrPSc andsPrPSc) (8). The neurotoxic form of PrP produced as a result ofprion replication is unknown (9, 10). Neuronal death can occurduring prion infection in the absence of detectable rPrPSc (11–13), suggesting that neurotoxic PrP is either sPrPSc or is distinctfrom PrPSc. In mice expressing anchorless prion protein, amyloidPrPSc can accumulate without clinical disease (14). In certainparadigms of experimental mouse scrapie, the time of onset ofneurological disease is distinct from the time when maximal in-fectivity and PrPSc levels are reached in the brain (15, 16). Col-lectively, these findings suggest that at least a substantial part ofneurotoxic PrP generated during prion infection is distinct fromPrPSc. Interestingly, in other misfolding neurodegenerative dis-eases, such as Alzheimer’s disease (AD), Parkinson disease (PD),and Huntington diseases, it has been shown that prefibrillar statesof involved proteins are toxic, rather than amyloid aggregates (9,17–20). Targeting neurotoxic PrP death pathways would be a newand powerful approach to therapy of prion diseases. Therefore, itis highly significant to define a highly neurotoxic form of PrP thatreproduces features of prion-induced neurodegeneration andcould be used as a model to study mechanisms of neuronal deathoccurring during prion infection. We set out to establish a mo-lecularly defined model for PrP-induced toxicity.

Besides model peptides, such as PrP106-126, 118–135, or 105–132 (21, 22), oligomers of recombinant PrP are neurotoxic (23, 24).We showed that PrP oligomers are not only toxic in vitro but alsoin vivo (25); however, they were poorly defined and containedoligomers of various sizes together with PrP monomers (25). Weimplemented a different strategy to identify neurotoxic PrP spe-cies. Instead of aiming at a particular type of PrP, we subjectedrecombinant PrP to denaturation and dilution refolding underconditions particularly suitable for disulfide bond-containing pro-teins and for the refolding of protein complexes (26, 27). Wefractionated the refoldedproduct on a sizing columnand examinedthe toxic properties of each PrP fraction on neuroblastoma cells.We found a highly neurotoxic PrP entity in the form ofmonomeric,α-helical PrP (called TPrP) harboring significant toxicity at a doseas low as 20 nM (0.5 μg/mL). TPrP was toxic for all three testedmurine neuronal cell types, but not formouse or humanfibroblasts.TPrP was also neurotoxic in vivo, showing about 10-times higherspecific activity than PrP oligomers. TPrP induced apoptosis,autophagy, and molecular hallmarks similar to those observed inprion-infected mouse brains. Although a PrP species equivalent toTPrP has yet to be identified in prion-infected brainmaterial, TPrPrepresents a highly neurotoxic form ofmisfolded prion protein thatmimics several features of prion pathogenesis and can serve asa model to study PrP-induced neurodegenerative mechanisms.

ResultsIdentification of TPrP, a Highly Toxic PrP Species. Recombinant full-length PrP from Escherichia coli was denatured, subjected todilution refolding, and the mixture of PrP conformers andaggregates of various sizes was separated by size-exclusionchromatography (SEC) (Fig. 1A). Fig. S1 shows the purity of PrPin inclusion bodies, which is comparable to that of previouslypublished PrP preparations (28). The two main bands of PrPconsistently observed throughout all fractions correspond to PrP23–231 and PrP 30–231, as described previously (29), and N-terminally truncated PrP was observed from fraction V93 toV101 (Fig. 1 B and G, and Fig. S2). Each 2-mL fraction wastested for toxicity on PK1 neuroblastoma cells (30). The fractionscorresponding to elution volumes V89, V91, and V93, collec-tively named fraction V89-93, were highly cytotoxic after 3 d, ina dose-dependent manner, starting from 0.5 μg/mL (Fig. 1D).Fractions V89 and V91 contained no or minute amounts oftruncated PrP, showing that toxicity was not because of PrPfragments (Fig. S2). Further analysis of fraction V89-93 ona Superdex 75 column showed that it contained one single majorPrP species that we called TPrP (Fig. 1C). Cell death wascharacterized by neuritic retraction and vacuolation (Fig. 1E).Soluble fractions of higher molecular weight than fraction V89-93, PrP aggregates, and the monomeric PrP species eluted infraction V109 [nontoxic PrP (NTPrP), which also resolves intoa single peak after further chromatographic separation] (Fig.1C), did not exhibit toxicity at concentrations up to 50 μg/mL.

Author contributions: M.Z. and C.I.L. designed research; M.Z., G.O., and G.F.S. performedresearch; M.Z., G.O., G.F.S., and C.I.L. analyzed data; and M.Z. and C.I.L. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1Present address: OPKO Health, Inc., Jupiter, FL 33458.2To whom correspondence should be addressed. E-mail: lasmezas@scripps.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1118090109/-/DCSupplemental.

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We took great care to ascertain that toxicity of fraction V89-93was PrP-specific. PK digestion eliminated toxicity, showing thattoxicity was not a result of a nonproteinaceous coeluted con-taminant (Fig. S3). As controls, we refolded in the same way thepolymeric Ig receptor pIGR D1-H6 and the Aβ42 peptide. Noneof the fractions of pIGR D1-H6 or Aβ42 peptide tested atconcentrations up to 5 and 50 μg/mL, respectively, exhibitedtoxicity on murine PK1 cells. However, one oligomeric Aβ42fraction induced dose-dependent neuronal death of human SK-N-SH cells (Fig. S4). We then reasoned that the most convincingdemonstration that the toxicity of TPrP was because of a con-former of PrP and not because of an adventitious contaminantwould be the conversion of a nontoxic PrP fraction into TPrP.We thus applied the refolding/fractionation process to PrPaggregates obtained after the first round of refolding. Theresulting TPrP (V86) was extremely pure (as seen on the silvergel) and exhibited toxicity similar to that of TPrP obtained fromrefolding of PrP from inclusion bodies (Fig. 1 F–H). Alterna-tively, we concentrated and freeze/thawed purified NTPrP andfractionated the resulting PrP on a Superdex 75 column. Afterthis procedure a very small peak corresponding to TPrPappeared at the expected elution volume. Collected “secondgeneration” TPrP exhibited toxicity similar to the “first genera-tion” TPrP (Fig. S5). This process demonstrated that the toxicityof TPrP was because of a specific PrP conformer.

TPrP Is an α-Helical PrP Monomer and Differs in Conformation fromNTPrP. Because conformation and hydrophobicity affect themobility of proteins in SEC, to determine the size of TPrP, wesubmitted two independent samples of purified TPrP to multi-angle light scattering analysis at the Analytical Services of WyattTechnology and at the Biophysics Resource of Keck Facility,Yale University, New Haven, CT. Both processes showed thatTPrP is a PrP monomer (Fig. S6). CD analyses on three differentpurified TPrP preparations revealed that TPrP is an α-helical PrPspecies (Fig. 2A), with higher α-helical content than NTPrP andOPrP (an oligomeric species partitioning in fraction V81) (Fig.1A). We then probed the conformation of TPrP using a con-

formation-dependent immunoassay. TPrP, similar to OPrP,exhibited reduced immunoreactivity to PrP mAb D18 and en-hanced resistance to guanidinium unfolding compared withNTPrP (Fig. 2B). Furthermore, TPrP was more resistant to mildPK digestion than NTPrP or even OPrP. Both TPrP and OPrPreleased a PK-resistant core at 0.1 μg/mL PK, but NTPrP did not(Fig. 2C). These data show that the conformation of TPrP isdifferent from that of NTPrP. CD and PK digestion showed thatTPrP also structurally differs from OPrP. Using protein mis-folding cyclic amplification according to the technique recentlydeveloped by Wang et al. (6), we showed that TPrP could beconverted into PrPSc in vitro equally well as NTPrP (Fig. 2D).

Similar Morphological and Molecular Hallmarks of TPrP-InducedNeuronal Damage and Prion-Infected Mouse Brains. TPrP-inducedneuronal damage resembled that observed in prion diseases.TPrP-treated PK1 cells underwent neuritic retraction and loss(Fig. 1 E and H) that have been described as early signs ofneuronal damage in prion diseases (31, 32) as well as apoptosis(Fig. 3A), a feature of prion pathology (11). TPrP inducedabundant neuronal vacuolation (Fig. 1 E and H), which is thehallmark of prion diseases (33) and has been recently linked toautophagy (34, 35). To examine if TPrP-induced vacuolation ismechanistically related to that occurring during prion diseases,we studied the expression levels of key molecular players of theautophagy pathway both in TPrP-exposed neuroblastoma cellsand in the brains of mice infected with three different prionstrains (Fig. 3B). We observed almost indistinguishable effects ofTPrP in cells and of scrapie infection in vivo as regards modu-lation of autophagolysosomal markers. The primary autopha-gosome marker LC3-II/I ratio was elevated, as well as thelysosomal marker Lamp2. Atg5 was unchanged in TPrP andscrapie-induced autophagy, and beclin-1 was reduced in bothcases. We noted an isolated discrepancy with mammalian targetof rapamycin (mTOR) being reduced at day 4 in TPrP-exposedcells and elevated in scrapie-infected mouse brains.

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Fig. 1. Neurotoxicity of PrP fraction TPrP incultured cells. (A) SEC of refolded PrP on aSuperdex 200 column. (B) A representativesample of the collected fractions V41-V121was analyzed on a denaturing gel (Coo-massie blue staining, Left) and by Westernblot (Right). Two-milliliter fractions werecollected. Numbers under the gels indicatestarting volume of each fraction. All fractionswere tested for neurotoxicity on PK1 cells.(C) Chromatograms of fraction V89-95 (blue)and fraction V109-113 (red) on a Superdex 75column. Chromatograms from separate SECruns were plotted on a single graph. Dashedlines show the fraction analyzed by multi-angle light scattering. (D) Dose-dependenttoxicity of fraction V89-95 (TPrP, blue). PK1cells were exposed to TPrP for 3 d and cellviability was measured in duplicate by theCell-Titer Glo assay. The effect of nontoxic,monomeric fraction V109-113 (NTPrP, red)is also shown. (E) Phase-contrast microscopyshowing the morphology of PK1 cells treatedwith 5 μg/mL of TPrP. Note the formation ofintracytoplasmic vacuoles, a typical featureof prion-infected brains and neuritic loss. (F–H) PrP aggregates can be refolded into TPrP.Fraction V86-100 contains TPrP. (F) SEC frac-tionation of denatured and refolded PrP ag-gregates on a Superdex 200 column. (G) Silver-stained SDS/PAGE gel from a representative sampling of collected fractions (Left) and PrP western-blot (Right).Numbers on each lane indicate starting volumes of collected fractions V41-62, V62-76, V76-86, V86-100, and V100-128, except “(1),” indicating denatured PrPbefore refolding. (H) Phase-contrast microscopy showing PK1 cells exposed for 3 d to various SEC fractions of refolded PrP aggregates (from G) containing5 μg/mL of PrP. Only fraction V86-100 exhibited neurotoxicity. (Magnification in E and H, 200×.)

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TPrP Is Toxic to Neuronal Cells but Not Fibroblasts. Mouse neuronalcells, including PK1, CAD5, and PrP−/−murine hippocampal neu-rons (Hpl3.4) (36), were susceptible to 2.5 μg/mL TPrP (Fig. S7).

In contrast, human and mouse fibroblasts (293T and LD9 cells,respectively) were not affected by up to 50 μg/mL TPrP (Fig. S7).These results suggest a neuron-specific susceptibility to TPrP.Fluorescently labeled TPrP (which retained its toxicity), was notdetectable above background levels inside fibroblastic cells, incontrast to the high levels found in neuronal cells (Fig. S8).

TPrP Is Metabolized Differently than NTPrP. Fluorescently labeledTPrP, NTPrP, and OPrP were all taken up by PK1 cells. Aftera single-day exposure, fluorescence associated with TPrP vanishedafter 1 d, that associated withOPrP after 2 d, but a label associatedwith NTPrP was found in the cells up to 5 d (Fig. S9). Intriguingly,as judged by fluorescence, TPrP and OPrP showed a differentpattern of accumulation than NTPrP. NTPrP was mostly seques-tered in smaller dot-like structures that did not coincide with TPrPor OPrP localization. TPrP largely colocalized with OPrP (Fig. 4).In addition, TPrP localized in vacuoles and at the cell surface inmembranous structures.We did not observe any increased toxicityas a result of NTPrP or OPrP cotreatment.

TPrP Is Highly Neurotoxic on Brain Slices and in Vivo. We tested thetoxicity of various SEC fractions on cultured mouse cerebellarbrain slices that are capable of replicating prions (37). TPrPinduced cell loss, resulting in shrinkage and partial destruction ofbrain slices in 6 d. NTPrP had no adverse effect on brain slices,whereas OPrP was toxic, but at a later time point and less so thanTPrP (Fig. S10). We then compared TPrP and OPrP toxicityafter subhippocampal stereotaxic injections into mouse brains:both induced dose-dependent apoptosis and death of pyramidalhippocampal neurons, as evidenced by the presence of shrunkencells harboring pyknotic nuclei, and neuronal loss (Fig. 5, yellowarrows). TPrP was about 10-times more toxic than OPrP (10-times more OPrP than TPrP was required to induce the sameextent of lesions). High doses of TPrP elicited lesions extendingto the dentate gyrus (Fig. 5A, green arrowhead), as well asastrogliosis and a bouquet of vacuoles in the hippocampus (Fig.5A, green arrow). PBS or NTPrP, injected into contralateralhemispheres as controls, failed to induce any brain lesions. Wehave not tested whether TPrP can trigger the formation of in-fectious prions after intracerebral injection.

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Fig. 2. TPrP is α-helical yet differs from NTPrP. (A) CD analysis of TPrP. TPrP(light blue line) is highly α-helical as inferred by the troughs at 208 and 222nm. NTPrP (pink line) has a slightly lower α-helical content than TPrP. OPrP(orange line) is partly disordered but retains some α-helical structure. (B)Probing the conformation of TPrP, NTPrP, and OPrP by conformation-de-pendent immunoassay. PrP was exposed to increasing amounts of GdnHCl;immunoreactivity to PrP mAb D18 was probed by direct ELISA. (C) TPrP ismore resistant to PK digestion than OPrP and NTPrP. A full-length TPrP bandis detectable up to 0.25 μg/mL of PK, and a PK-resistant core (arrow) is gen-erated by 0.1 μg/mL of PK. A full-length OPrP band is detectable up to 0.1 μg/mL of PK, and a PK-resistant core is generated. No PK-resistant NTPrP core isgenerated. PrP concentration was 100 μg/mL in all preparations. All samples(equal protein amounts) were loaded on a single gel. Because of the differ-ence in immunoreactivity between NTPrP and TPrP, as well as OPrP, theWestern blot membrane was divided in three to allow adjustment of expo-sure times. Different exposure times did not change findings. (D) TPrP andNTPrP can be converted into PrPSc by protein misfolding cyclic amplificationwith similar efficiency. F, frozen control seed consisting of de novo generatedPK-resistant recombinant PrP (6); A, amplified TPrP or NTPrP.

β

A BFig. 3. TPrP induces apopto-sis, autophagy, and the samemolecular signature as thatinduced in brain by in vivoscrapie infection. (A) TPrP-treated cells were labeled foractivated caspases (green,Top Right), activated caspase8 (green, Middle Right), andcaspase 9 (red, Bottom Left).Control indicates untreatedcells labeled for activatedcaspases. (Bottom Right) Amerge of caspase 8 and 9 la-beling. The orange-to-yellowsignal shows that both apo-ptotic pathways are activatedin the same cells, and even inthe same subcellular loca-tions. Some caspase labelingis clearly found in vacuoles(yellow arrows) but somevacuoles are devoid of cas-pase labeling (blue arrows).Cell nuclei were labeled byHoechst staining (blue). (B)Western blots showing the effects of TPrP treatment of cells and scrapie infection of brain on markers of the autophagy pathway. (Left) Analyses fromPK1 cells at days 2, 3, and 4 of TPrP treatment and from control cells at similar time points are shown. (Right) Analyses of three individual healthy mousebrains and of pooled brain homogenates from scrapie-sick mice (after infection with either RML, 22L, or ME7 prions) are shown. GAPDH serves as theloading control. Arrows show changes induced by TPrP treatment and scrapie infection. (Magnification, 200×.)

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DiscussionPrecisely defining neurotoxic species in protein misfolding dis-eases is a major challenge and the gaps of knowledge in thisregard prevent the development of targeted therapeutic strate-gies. In recent years, the concept has developed that β-sheet–structured oligomeric species of amyloidogenic proteins are theculprit, because toxicity has been found associated with smallmolecular-weight aggregates of several such proteins. Several

studies, including one of ours, showed that oligomers of the prionprotein are neurotoxic (23–25). However, highly α-helical mo-nomeric intermediates in amyloid formation have been discov-ered for amyloidogenic proteins, such as Aβ, α-synuclein, and isletamyloid polypeptide (IAPP) involved in the pathogenesis of AD,PD, and type II diabetes, respectively (38–41). Studies of PrPdenaturation/renaturation have shown that PrP refolds into anα-helical PrP monomer followed by dimerization and

Fig. 4. TPrP colocalizes with OPrP but notNTPrP in PK1 cells. TPrP, OPrP, and NTPrP werelabeledwith either Alexa-488 (green) or Alexa-555 (red), as indicated on each picture, andcells were exposed to two of them in combi-nation for 3 d (5 μg/mL). (Upper) TPrP andOPrPshow a largely overlapping intracellular local-ization (yellow areas in “merge” panel). Ad-ditionally, TPrP localized in vacuoles and at thecell surface in membranous structures (bluearrows), which were preferentially associatedwith neurites when these were still present(red arrows). (Lower) NTPrP does not colocalizewith TPrP. NTPrP is partly sequestered in smalldot-like structures (white arrows) but not TPrP.(Magnification, 400×.)

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Fig. 5. High toxicity of TPrP in vivo. TPrP exhibits higher toxicity in brain than OPrP. TPrP (A) or OPrP (B) was injected stereotaxically above the hippocampusof C57BL/6J mice. Mouse brains were examined histopathologically after Nissl staining, 5 d after the injection. Scale bars indicate the extent of damaged area.Note the complete loss of pyramidal neurons in the center of the damaged area for the higher TPrP dose (1.4 μg). Yellow arrows indicate shrunken apoptoticneurons and neuronal loss. The green arrow shows a bouquet of small vacuoles, and the green arrowhead shows shrunken apoptotic neurons in the dentategyrus after high dose TPrP injection. (Upper) Magnification, 5×; (Lower) magnification, 10×. (Scale bars, 100 μm.).

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conformational transition to β-sheeted oligomers (42). It has beenproposed that α-helix formation would lead to a high local con-centration of an aggregation-prone sequence, in turn promotingintermolecular β-sheet formation (43). Consistent with this hy-pothesis, acceleration of amyloid formation is correlated with theamount of helical IAPP (44, 45). Binding to cellular membranesinduces the formation of α-helices in Aβ, α-synuclein, and IAPP(40, 46). Interestingly, the neurotoxicity of cyPrP, a cytoplasmicform of PrP, correlates with its interaction with lipid membranesin transgenic mouse brains (47, 48). Moreover, preventing theformation of β-structure by mutagenesis in the N-terminal regionof α-synuclein increases its neurotoxicity in PD models (49). Thequestion has been asked as to whether any of these α-helical andpresumably early folding intermediates can represent toxic spe-cies amenable to therapeutic targeting (39). A peptidomimeticapproach targeting α-helical IAPP intermediates inhibited fiberformation under lipid-catalyzed conditions and attenuated IAPP-induced toxicity (50). Herein, we provide unique experimentalevidence that a highly α-helical amyloidogenic protein is stronglyneurotoxic.TPrP appeared as the most toxic recombinant PrP species

hitherto generated, with activity in the nanomolar range [0.5 μg/mL; (i.e., 20 nM)], whereas β-sheeted forms of PrP or fragmentsthereof are toxic in themicromolar range (24, 25, 51–53). TPrPwashighly neurotoxic on various types of neuronal cells, on cerebellarbrain slices, and in mouse brains. OPrP was only toxic on brainslices and in vivo, where it was about 10-times less toxic than TPrP.TPrP differed from NTPrP, its nontoxic monomeric counterpart,by its conformation, as shown by its different immunoreactivityusing the conformation-dependent immunoassay, by its higherresistance to mild PK digestion, and by its different SEC elutionprofile. Interestingly, the fact that TPrP exhibits higher PK re-sistance than NTPrP and OPrP, despite higher α-helical contentshows that increased PK-resistance does not necessarily go hand-in-hand with the acquisition of higher β-sheet content. It will be ofgreat interest to determine the structural differences betweennative and toxic α-helical PrP. TPrP andNTPrPwere equally goodsubstrates for in vitro conversion. Therefore, even though wecannot exclude that a species equivalent to TPrP might be pro-duced independently of PrPSc during prion infection, itmight be anearly on-pathway intermediate generated during the structuraltransition of PrP into PrPSc.We found that TPrP is toxic even in the absence of endoge-

nous neuronal PrP expression. This finding is consistent with ourprevious in vitro and in vivo findings (25); it does not contradictearlier studies reporting that PrPSc deposits do not induce lesionsin PrP knock-out mouse brains (54) and that postnatal PrP de-pletion prevents scrapie-disease (55), as in both cases absence ofPrP expression precludes the formation of newly misfolded toxicPrP. Interestingly, Strittmatter and colleagues (56, 57) discov-ered a role for cellular PrP as a mediator of Aβ oligomer toxicitythat has been extended to other β-sheet–rich protein conformers(58), showing the existence of another neurotoxic pathway.Toxic PrP has yet to be isolated from prion-infected mouse

brains. This is an extremely difficult task because, although TPrPis a stable PrP species in our experimental conditions, toxic PrPmight be short-lived in the context of prion replication; moreoverwe show that toxic PrP (TPrP and OPrP) is rapidly disposed of byPK1 cells, much more so than nontoxic PrP. The temporal andtopological dissociation between neuronal death and PrPSc ac-cumulation evidenced in certain models of prion diseases (11–16) suggests that at least one major component of PrP-inducedtoxicity is different from PrPSc. PrPSc preparations from prion-infected brains were found to exhibit toxicity in cultured cells (59,60). PrPSc by itselfmight also exert some toxicity, or neurotoxic PrPmight copurify with PrPSc after sarkosyl-induced PrP aggregationand centrifugation. TPrP recapitulates the specific morphologicaland molecular phenotype of cell death occurring during priondiseases: extensive vacuolation, apoptosis, and autophagy (11, 33–35). We therefore probed molecular markers involved in theautophagolysosomal degradation pathway in TPrP-exposed

neuronal cells andmouse brains infected by three different scrapiestrains (22L,RML, andME7). In both cells and brains, the primaryautophagosome marker LC3-II/I ratio was elevated as well as thelysosomal marker Lamp2, indicating induction of autophagy orblock in autophagy execution. Atg5 was unchanged, in contrast to,for example, adenovirus-induced autophagy (61) but similar tobrains of patients andmousemodels of ADand α-synucleinopathy(62). Masliah and colleagues showed that lentiviral beclin-1overexpression reduces amyloid pathology in APP transgenicmice, as well as α-synuclein accumulation and degenerate pa-thology in α-synuclein overexpression models in vitro and in vivo;these data suggest a role of beclin-1 in autophagy induction andpossibly targeting excess misfolded protein to the autophagypathway (63, 64). Beclin-1 was reduced in TPrP-treated neuronsand in scrapie-infected brains, suggesting a deleterious exhaustionof this early player of the autophagic pathway. Interestingly,beclin-1 is also reduced in the brains of AD patients (62, 64), butnot in dementia with Lewy Bodies related to α-synuclein de-position (62), emphasizing the specificity of molecular alterationsoccurring in different protein-misfolding neurodegenerative dis-eases. In summary, the molecular signature of autophagy in-duction was similar after TPrP-induced toxicity and prion-inducedtoxicity, with the notable exception of mTOR. This finding is notsurprising, given the multifaceted roles of mTOR that are notlimited to autophagy but include transcription activation, whichcertainly takes place as part of the gliotic response in prion-infected brains. Of note, even though alterations of the autophagypathway are a salient feature of TPrP-induced neuronal death andscrapie-infected brains, the role of autophagy in the neurode-generative process remains to be determined.It might be that several toxic PrP species are generated during

prion replication. TPrP might be the equivalent of one of them.Because of TPrP’s exquisite, neuron-specific toxicity and thesimilarities between TPrP- and prion-induced toxicity, we proposeTPrP as a model to study PrP-induced neurodegeneration.Toxic PrP species (both TPrP and OPrP) are compartmental-

ized in neurons differently and are cleared more rapidly thannontoxic PrP.TPrPmight be secreted fromcells.Alternatively, ourdata showing elevated LC3II/I ratios are compatible with in-duction of autophagy and rapid degradation of TPrP. Although,obviously, TPrP and PrPSc are different entities, it is noteworthythat PrPSc degradation is accelerated by autophagy activation (65,66). Further studies aiming at understanding the differences be-tween NTPrP and TPrP cellular processing will shed light on thecellular mechanisms of TPrP-induced neurotoxicity.The present study challenges the prevailing concept that neu-

ronal damage is solely or mainly linked to the toxicity of β-sheetPrP oligomers. Although a role of β-sheet oligomers in prionpathogenesis is not ruled out by our study, the identification ofa highly toxic α-helical PrP monomer opens up a new chapter inthe understanding of prion-induced neurodegeneration. Finally,our findings foster the tentative proposal that highly helicalprotein conformers might be involved in cellular toxicity not onlyin prion diseases but also in other protein misfolding diseases.

Materials and MethodsSI Materials and Methods contains a detailed description of the productionand SEC fractionation of recombinant proteins. It also describes PrP labeling,conversion of PrP aggregates or NTPrP into TPrP, CD analysis, multianglelight scattering, protein misfolding cyclic amplification, PK digestion, and gelelectrophoresis and Western blotting. Details are given about cell culture,cell treatment, immunocytochemistry, cell viability assays, brain slice cul-tures, as well as in vivo toxicity assay and mouse infections.

ACKNOWLEDGMENTS. We thankDr. JiyanMa for the gift of the recombinantproteinase K-resistant prion protein seed and for his advice on the proteinmisfolding cyclic amplification procedure using recombinant prion protein;Dr. Nicole Salès for her support with immunocytochemistry; Dr. Michelle Chenfor her help with multiangle light scattering (MALS) and interpretation ofMALS data; Dr. Alexandra Sherman for preparing brain slices; and Dr. Guil-laume Mousseau for assistance with circular dichroism.

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1. Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216:136–144.

2. Pan KM, et al. (1993) Conversion of alpha-helices into beta-sheets features in theformation of the scrapie prion proteins. Proc Natl Acad Sci USA 90:10962–10966.

3. Legname G, et al. (2004) Synthetic mammalian prions. Science 305:673–676.4. Deleault NR, Harris BT, Rees JR, Supattapone S (2007) Formation of native

prions from minimal components in vitro. Proc Natl Acad Sci USA 104:9741–9746.

5. Kim JI, et al. (2010) Mammalian prions generated from bacterially expressedprion protein in the absence of any mammalian cofactors. J Biol Chem 285:14083–14087.

6. Wang F, Wang X, Yuan CG, Ma J (2010) Generating a prion with bacterially expressedrecombinant prion protein. Science 327:1132–1135.

7. Weissmann C (1991) A ‘unified theory’ of prion propagation. Nature 352:679–683.8. Safar J, et al. (1998) Eight prion strains have PrP(Sc) molecules with different con-

formations. Nat Med 4:1157–1165.9. Caughey B, Lansbury PT (2003) Protofibrils, pores, fibrils, and neurodegeneration:

Separating the responsible protein aggregates from the innocent bystanders. AnnuRev Neurosci 26:267–298.

10. Aguzzi A, Sigurdson C, Heikenwaelder M (2008) Molecular mechanisms of prionpathogenesis. Annu Rev Pathol 3:11–40.

11. Lasmézas CI, et al. (1997) Transmission of the BSE agent to mice in the absence ofdetectable abnormal prion protein. Science 275:402–405.

12. Gambetti P, Parchi P, Petersen RB, Chen SG, Lugaresi E (1995) Fatal familial insomniaand familial Creutzfeldt-Jakob disease: Clinical, pathological and molecular features.Brain Pathol 5:43–51.

13. Manson JC, et al. (1999) A single amino acid alteration (101L) introduced into murinePrP dramatically alters incubation time of transmissible spongiform encephalopathy.EMBO J 18:6855–6864.

14. Chesebro B, et al. (2005) Anchorless prion protein results in infectious amyloid diseasewithout clinical scrapie. Science 308:1435–1439.

15. Büeler H, et al. (1994) High prion and PrPSc levels but delayed onset of disease inscrapie-inoculated mice heterozygous for a disrupted PrP gene. Mol Med 1:19–30.

16. Sandberg MK, Al-Doujaily H, Sharps B, Clarke AR, Collinge J (2011) Prion propagationand toxicity in vivo occur in two distinct mechanistic phases. Nature 470:540–542.

17. Kayed R, et al. (2003) Common structure of soluble amyloid oligomers implies com-mon mechanism of pathogenesis. Science 300:486–489.

18. Lesné S, et al. (2006) A specific amyloid-beta protein assembly in the brain impairsmemory. Nature 440:352–357.

19. Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: lessonsfrom the Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Biol 8:101–112.

20. Winner B, et al. (2011) In vivo demonstration that alpha-synuclein oligomers are toxic.Proc Natl Acad Sci USA 108:4194–4199.

21. Forloni G, et al. (1993) Neurotoxicity of a prion protein fragment.Nature 362:543–546.22. Haïk S, et al. (2000) Neurotoxicity of the putative transmembrane domain of the

prion protein. Neurobiol Dis 7(6 Pt B):644–656.23. Kazlauskaite J, et al. (2005) An unusual soluble beta-turn-rich conformation of prion

is involved in fibril formation and toxic to neuronal cells. Biochem Biophys ResCommun 328:292–305.

24. Novitskaya V, Bocharova OV, Bronstein I, Baskakov IV (2006) Amyloid fibrils ofmammalian prion protein are highly toxic to cultured cells and primary neurons. J BiolChem 281:13828–13836.

25. Simoneau S, et al. (2007) In vitro and in vivo neurotoxicity of prion protein oligomers.PLoS Pathog 3:e125.

26. Garboczi DN, Hung DT, Wiley DC (1992) HLA-A2-peptide complexes: Refolding andcrystallization of molecules expressed in Escherichia coli and complexed with singleantigenic peptides. Proc Natl Acad Sci USA 89:3429–3433.

27. Buchner J, Rudolph R (1991) Renaturation, purification and characterization of re-combinant Fab-fragments produced in Escherichia coli. Biotechnology (N Y) 9:157–162.

28. Makarava N, Baskakov IV (2008) Expression and purification of full-length recombi-nant PrP of high purity. Methods Mol Biol 459:131–143.

29. Ostapchenko VG, Makarava N, Savtchenko R, Baskakov IV (2008) The polybasic N-terminal region of the prion protein controls the physical properties of both thecellular and fibrillar forms of PrP. J Mol Biol 383:1210–1224.

30. Klöhn PC, Stoltze L, Flechsig E, Enari M, Weissmann C (2003) A quantitative, highlysensitive cell-based infectivity assay for mouse scrapie prions. Proc Natl Acad Sci USA100:11666–11671.

31. Fuhrmann M, Mitteregger G, Kretzschmar H, Herms J (2007) Dendritic pathology inprion disease starts at the synaptic spine. J Neurosci 27:6224–6233.

32. Brown D, Belichenko P, Sales J, Jeffrey M, Fraser JR (2001) Early loss of dendritic spinesin murine scrapie revealed by confocal analysis. Neuroreport 12:179–183.

33. Fraser H (1976) The pathology of natural and experimental scrapie. Slow Virus Dis-eases of Animals and Man, ed Kimberlin RH (North-Holland, New York), pp 267–305.

34. Liberski PP, BrownDR, Sikorska B, Caughey B, BrownP (2008) Cell death and autophagyin prion diseases (transmissible spongiform encephalopathies). Folia Neuropathol 46:1–25.

35. Heiseke A, Aguib Y, Schatzl HM (2010) Autophagy, prion infection and their mutualinteractions. Curr Issues Mol Biol 12:87–97.

36. Sakudo A, Onodera T, Ikuta K (2007) Prion protein gene-deficient cell lines: Powerfultools for prion biology. Microbiol Immunol 51:1–13.

37. Falsig J, et al. (2008) A versatile prion replication assay in organotypic brain slices. NatNeurosci 11:109–117.

38. Kirkitadze MD, Condron MM, Teplow DB (2001) Identification and characterization ofkey kinetic intermediates in amyloid beta-protein fibrillogenesis. J Mol Biol 312:1103–1119.

39. Abedini A, Raleigh DP (2009) A role for helical intermediates in amyloid formation bynatively unfolded polypeptides? Phys Biol 6:015005.

40. Hebda JA, Miranker AD (2009) The interplay of catalysis and toxicity by amyloid inter-mediates on lipid bilayers: Insights from type II diabetes.AnnuRev Biophys 38:125–152.

41. Anderson VL, Ramlall TF, Rospigliosi CC, Webb WW, Eliezer D (2010) Identification ofa helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation. ProcNatl Acad Sci USA 107:18850–18855.

42. Jansen K, et al. (2001) Structural intermediates in the putative pathway from thecellular prion protein to the pathogenic form. Biol Chem 382:683–691.

43. Abedini A, Raleigh DP (2009) A critical assessment of the role of helical intermediatesin amyloid formation by natively unfolded proteins and polypeptides. Protein EngDes Sel 22:453–459.

44. Knight JD, Hebda JA, Miranker AD (2006) Conserved and cooperative assembly ofmembrane-bound alpha-helical states of islet amyloid polypeptide. Biochemistry 45:9496–9508.

45. Saraogi I, et al. (2010) Synthetic alpha-helix mimetics as agonists and antagonists ofislet amyloid polypeptide aggregation. Angew Chem Int Ed Engl 49:736–739.

46. Eliezer D, Kutluay E, Bussell R, Jr., Browne G (2001) Conformational properties ofalpha-synuclein in its free and lipid-associated states. J Mol Biol 307:1061–1073.

47. Wang X, et al. (2009) Cytoplasmic prion protein induces forebrain neurotoxicity. Bi-ochim Biophys Acta 1792:555–563.

48. Wang X, Wang F, Arterburn L, Wollmann R, Ma J (2006) The interaction betweencytoplasmic prion protein and the hydrophobic lipid core of membrane correlateswith neurotoxicity. J Biol Chem 281:13559–13565.

49. Karpinar DP, et al. (2009) Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in Parkinson’s disease models. EMBO J 28:3256–3268.

50. Hebda JA, Saraogi I, MagzoubM, Hamilton AD,Miranker AD (2009) A peptidomimeticapproach to targeting pre-amyloidogenic states in type II diabetes. Chem Biol 16:943–950.

51. Huang L, et al. (2010) Macromolecular crowding converts the human recombinantPrPC to the soluble neurotoxic beta-oligomers. FASEB J 24:3536–3543.

52. Villa V, et al. (2006) Characterization of the proapoptotic intracellular mechanismsinduced by a toxic conformer of the recombinant human prion protein fragment 90-231. Ann N Y Acad Sci 1090:276–291.

53. Sanghera N, Wall M, Vénien-Bryan C, Pinheiro TJ (2008) Globular and pre-fibrillarprion aggregates are toxic to neuronal cells and perturb their electrophysiology. Bi-ochim Biophys Acta 1784:873–881.

54. Brandner S, et al. (1996) Normal host prion protein necessary for scrapie-inducedneurotoxicity. Nature 379:339–343.

55. Mallucci G, et al. (2003) Depleting neuronal PrP in prion infection prevents diseaseand reverses spongiosis. Science 302:871–874.

56. Laurén J, Gimbel DA, Nygaard HB, Gilbert JW, Strittmatter SM (2009) Cellular prionprotein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature457:1128–1132.

57. Gimbel DA, et al. (2010) Memory impairment in transgenic Alzheimer mice requirescellular prion protein. J Neurosci 30:6367–6374.

58. Resenberger UK, et al. (2011) The cellular prion protein mediates neurotoxic signallingof β-sheet-rich conformers independent of prion replication. EMBO J 30:2057–2070.

59. Hetz C, Russelakis-Carneiro M, Maundrell K, Castilla J, Soto C (2003) Caspase-12 andendoplasmic reticulum stress mediate neurotoxicity of pathological prion protein.EMBO J 22:5435–5445.

60. Hetz C, et al. (2005) The disulfide isomerase Grp58 is a protective factor against prionneurotoxicity. J Neurosci 25:2793–2802.

61. Rodriguez-Rocha H, et al. (2011) Adenoviruses induce autophagy to promote virusreplication and oncolysis. Virology 416:9–15.

62. Crews L, et al. (2010) Selective molecular alterations in the autophagy pathway in pa-tients with Lewy body disease and in models of alpha-synucleinopathy. PLoS ONE 5:e9313.

63. Spencer B, et al. (2009) Beclin 1 gene transfer activates autophagy and amelioratesthe neurodegenerative pathology in alpha-synuclein models of Parkinson’s and Lewybody diseases. J Neurosci 29:13578–13588.

64. Pickford F, et al. (2008) The autophagy-related protein beclin 1 shows reduced ex-pression in early Alzheimer disease and regulates amyloid beta accumulation in mice.J Clin Invest 118:2190–2199.

65. Heiseke A, Aguib Y, Riemer C, Baier M, Schätzl HM (2009) Lithium induces clearanceof protease resistant prion protein in prion-infected cells by induction of autophagy. JNeurochem 109:25–34.

66. Aguib Y, et al. (2009) Autophagy induction by trehalose counteracts cellular prioninfection. Autophagy 5:361–369.

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