Inherent toxicity of aggregates implies acommon mechanism for proteinmisfolding diseasesMonica Bucciantini*†, Elisa Giannoni*†, Fabrizio Chiti*§†, Fabiana Baroni*, Lucia Formigli‡, Jesus Zurdo§, Niccolo Taddei*,Giampietro Ramponi*, Christopher M. Dobson§ & Massimo Stefani*

* Dipartimento di Scienze Biochimiche, Viale Morgagni 50; ‡ Dipartimento di Anatomia, Istologia e Medicina legale, Viale Morgagni 85,Universita degli Studi di Firenze, 50134 Firenze, Italy§ Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK† These authors contributed equally to this work

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A range of human degenerative conditions, including Alzheimer’s disease, light-chain amyloidosis and the spongiformencephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. Ithas been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can beformed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3 0-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in theaggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidencethat avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in theorigins of this family of protein deposition diseases.

An increasing body of evidence suggests that amyloid formation isthe fundamental cause of protein deposition diseases1,2. The natureof the pathogenic species and the mechanism by which the aggrega-tion process results in cell damage are, however, the subject ofintense debate3 – 10. In systemic non-neurological diseases theaccumulation of large quantities (sometimes kilograms) of aggre-gated species within a variety of organs and tissues may itself be themajor cause of clinical symptoms11. In other cases, particularly thedegenerative neurological diseases, it appears likely that impairmentof cellular function is directly linked to the interaction of proteinaggregates with cellular components12,13. One specific indicator ofthe significance of aggregate formation in pathological conditions isthe evidence for a high variability in the age of disease onset14, anobservation that has recently been linked to the evidence thataggregates form by nucleation15. Further clues to the molecularbasis of amyloid diseases and the biological significance of proteinaggregation have been provided by recent observations that a rangeof proteins not associated with amyloid diseases are able toaggregate in vitro into fibrils indistinguishable from those foundin pathologic conditions16 – 19. This finding has led to the proposalthat aggregation can be viewed as a general property of polypeptidechains rather than one restricted to a small number of sequences2. Inthe light of this conclusion we have now examined the effects on cellviability of aggregated species produced in vitro from two suchproteins.

The SH3 domain from bovine phosphatidyl-inositol-3 0-kinase(PI3-SH3) and the N-terminal (‘acylphosphatase-like’) domain ofthe E. coli HypF protein (HypF-N) are two examples of smallglobular proteins that can form fibrillar aggregates in vitro underappropriate conditions17,20. Evidence that the aggregates formedfrom PI3-SH3 and HypF-N can be classified as amyloid fibrils hasbeen obtained from electron microscopy, specific tests such asCongo red and thioflavine T binding and, in the case of PI3-SH3,X-ray diffraction17,20. The heterogeneous nature of the in vitroaggregation process is a potential difficulty in experiments aimedat probing the nature of aggregate pathogenicity; this problem canhinder the identification of the particular species responsible for

any observed toxicity. We have found, however, that highly homo-geneous populations of various types of aggregates of PI3-SH3 orHypF-N can be obtained by incubating either protein under welldefined solution conditions for specific lengths of time. Thesesequentially and structurally unrelated proteins are therefore excep-tionally favourable systems for investigating any inherent cytotox-icity of specific types of proteinaceous aggregates.

Cytotoxicity of PI3-SH3 aggregatesIncubation of PI3-SH3 in either a 50-mM acetate buffer solution,pH 5.5, containing 25% (v/v) trifluoroethanol (TFE) or a H2O/HClmixture, pH 2.0, results in the formation of granular or fibrillaraggregates, respectively (Fig. 1). Both types of aggregates enhancethe fluorescence of thioflavin T (ThT) by factors of more than 30,indicating the presence of amyloid-like features within these struc-tures (data not shown). Examination by transmission electronmicroscopy (TEM) reveals that the aggregates formed rapidly atpH 5.5 in solutions containing TFE appear as granules 4–200 nm indiameter (Fig. 1d–f) without any detectable fibrillar species. Pro-longed incubation at pH 2.0, however, yields 7–13-nm-wide fibrilsin the absence of any detectable granular aggregates (Fig. 1b); thewidth of the fibrils observed here is characteristic of the ex vivoamyloid fibrils extracted from patients suffering from variousforms of amyloid disorders21. Some fibrils of this type formedfrom PI3-SH3 have been shown to consist of a double helicalarrangement of two pairs of ribbon-like protofilaments woundaround a hollow core22. In the samples analysed here, such fibrilsare occasionally found to split into two 6–7-nm-wide fibrils or toassociate further in a parallel fashion to produce 23- or 33-nm-widesheet-like fibrils, also in agreement with previous findings22.

The cytotoxicity of the two types of aggregates formed in suchexperiments was examined by adding aliquots of the aggregates, at arange of final protein concentrations (see Fig. 1 legend), to cellculture media. Aggregate cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)reduction inhibition assay, a standard indicator of cell physiologicalstress thought to be related to changes in intracellular trafficking,

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particularly in the pathway of exocytosis23,24. The experimentsreveal that the highly structured PI3-SH3 fibrils formed by pro-longed incubation at pH 2 do not significantly modify MTTreduction in either NIH-3T3 or PC12 cells even at the highestconcentration tested (Fig. 1a). The presence of the granular aggre-gates formed after short incubation periods, however, significantlyreduces cell viability (Fig. 1c, P , 0:01 at PI3-SH3 concentrationsabove 1 mM). No significant decrease of MTT reduction wasdetected when the cells were exposed either to 20 mM native PI3-SH3 or to the buffer solutions used to form the aggregates in theabsence of added protein (Fig. 1).

Cytotoxicity of HypF-N aggregatesIn a second series of experiments, the toxicity of aggregates formedby the protein HypF-N was examined. HypF-N was incubated at0.3 mg ml21 concentration in 50 mM acetate buffer, pH 5.5, in thepresence of 30% (v/v) TFE at room temperature; aliquots of thissolution were withdrawn at regular time intervals for examinationby TEM and for ThT and cytotoxicity assays on cultured NIH-3T3and PC12 cells. Under these conditions, aggregates develop withinminutes; these initial aggregates exhibit both a CD (circular dichro-ism) spectrum indicative of b-sheet structure and an enhancementof the ThT fluorescence by over 50 times relative to that of thesoluble native domain. At this stage of incubation, however, theaggregates appear completely non-fibrillar and non-granular inTEM images (Fig. 2b), whereas after 48 h of incubation somefibrillar character is clearly evident (Fig. 2c). The latter aggregateshave a width of about 4–8 nm and are extremely short (typically25–60 nm), resembling the protofibrils observed with other pro-

teins at relatively early stages of fibril formation25. The ends of theseaggregates appear disordered, suggesting they are in the process ofundergoing a transition from amorphous to fibrillar structures.After about 20 days, aggregates of this type can no longer be detectedin the samples; at this time all the aggregates appear as longunbranched fibrils with widths of either 3–5 or 7–9 nm (Fig. 2d,e), characteristic of constituent protofilaments and mature amyloidfibrils, respectively20.

Addition to the cell medium of the aggregates formed after a 6 hincubation period resulted in a marked decrease in the MTTreduction by both NIH-3T3 (Fig. 2a) and PC12 cells (data notshown). This decrease is statistically highly significant at all proteinconcentrations tested (0.04–20 mM; P , 0:01 at 0.04 mM) withrespect to controls performed by incubating the same cells with20 mM HypF-N in its soluble form or with the buffer solutions in theabsence of protein. A series of experiments revealed that aggregatetoxicity depends on protein concentration and initially increaseswith the length of exposure to the conditions that result inaggregation, reaching a maximum after 48 h (Fig. 2a). Incubatingcells with protein aggregates formed at this time results in aninhibition of MTT reduction ranging from 20% at the lowestprotein concentration used here (0.04 mM) to 70% at the highestprotein concentration (20 mM) with respect to the control exper-iments. In addition to impairing cellular function, HypF-N aggre-gates were also found to lead to cell death, as revealed by the trypanblue internalization test (Fig. 3). The results show that the rate of cellmortality increases until it reaches nearly 40% at the highest proteinconcentrations, indicating that the cellular dysfunction shown bythe MTT assay leads to cell death. Addition to the cell medium of

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Figure 1 PI3-SH3 aggregation and cytotoxicity. a, c, Cytotoxic effect of fibrillar (a) and

granular (c) PI3-SH3 aggregates on NIH-3T3 cells. Reported values are those after

treatment with 20 mM native protein (Native), with cell medium containing an aliquot of the

solution where aggregates were formed (HCl or TFE) or with aggregates at the indicated

protein concentrations (PI3-SH3). Values are relative to those of control cells treated with

complete medium alone. b, d– f, Electron micrographs showing fibrillar (b) and granular

(d– f) aggregates formed from PI3-SH3. Labels 1 and 2 indicate large granules and

clusters of small granules, respectively (e, f).

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protein samples incubated for times longer than 48 h, however,resulted in a progressive decrease of cell impairment that correlates,at least qualitatively, with the increase in the proportion of maturefibrils relative to other aggregates in the protein samples. Even atthe highest protein concentrations analysed, MTT reductionapproaches the value of the controls when the cells were exposedto protein samples incubated for 21 days (Fig. 2a), conditions whereonly mature fibrils are evident in the TEM micrographs. A lack oftoxicity was also found when cells were exposed to well definedamyloid fibrils formed at low pH (50 mM citric acid, pH 3.0) (datanot shown).

The origins of aggregate toxicityThe results of the present study indicate that aggregates formedfrom PI-SH3 and HypF-N can be substantially cytotoxic. In bothcases, the cytotoxicity was found to depend on the supramolecularorganization of the amyloid aggregates and is much more pro-nounced for the rapidly formed non-fibrillar aggregates than forthe highly organized fibrillar structures. The results for these twoproteins, neither of which is disease-related, are very similar to thosereported for the disease-associated fusogenic prion protein fragment,for a-synuclein, for Ab(1–42) and for transthyretin3 – 10,26. Remark-ably, the levels of cell impairment induced by the most toxic speciesfound in the present study are comparable to those of the highlytoxic aggregates formed from Ab(1–42) (ref. 27). The data, there-fore, suggest that impairment of cell viability by protein aggregatesof the type that can subsequently form amyloid fibrils could be ageneral phenomenon and not simply a specific property of the smallnumber of polypeptides associated with clinically recognized pro-tein deposition diseases. This result is of particular significance inthe light of the recent conclusion that the ability to form highly

ordered amyloid fibrils is itself a generic property of proteins17 – 19.In addition, the mature fibrils of the proteins we tested appear tobe essentially harmless to cells, providing an explanation forprevious data indicating that well defined amyloid fibrils formedby a synthetic peptide are not cytotoxic27 and reinforcing thefindings on the cytotoxicity of Ab, a-synuclein and transthyretinpre-fibrillar assemblies both in cultured cells and in transgenicmice3 – 5,9,10,26,10,28.

The results reported in this paper, therefore, provide evidencethat protein aggregates not associated with disease can be inherentlycytotoxic and result in substantial impairment of cellular function

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protofibrillar and fibrillar HypF-N aggregates. Cell viability was checked by the MTT

inhibition reduction test, after addition to the cell medium of either 20 mM native protein

(black circles) or different concentrations of protein incubated in TFE: 20 mM (blue), 5 mM

(red), 1 mM (green), 0.2 mM (purple) and 0.04 mM (cyan). Values are relative to control

cells treated with complete medium alone. The electron micrographs show amorphous

aggregates of HypF-N, formed after 6 h incubation (b), amorphous aggregates developing

into fibrils after 48 h incubation (c) and mature amyloid protofilaments (d) and fibrils (e)

after 20 days incubation.

Figure 3 Percentage of cell deaths induced by 48-h-aged HypF-N aggregates at different

protein concentrations. Solid bars refer to aggregated protein, dotted bars to control

experiments performed in the presence of soluble protein (see Methods for details).

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or even cell death. There is a considerable debate as to whether fullyformed mature amyloid fibrils or rapidly formed aggregates thatprecede their formation are the primary pathogenic species respon-sible for the onset of disease3 – 10. Although there is evidence for thetoxicity of mature fibrils in some amyloid diseases, the data reportedhere support previous suggestions that, at least in some cases, thenon-fibrillar aggregates that precede formation of mature amyloidfibrils may be the primary toxic species3 – 5,9,10,26,10,28. This toxicityis likely to arise because in these early aggregates hydrophobic side-chains and other regions of the polypeptide chain will be muchmore accessible than in the fully formed mature fibrils. Indeed, thelatter are often found to be remarkably inert, for example in theirresistance to proteolysis and degradation29.

The inherent cytotoxicity of the aggregates formed by the twonon-disease-related proteins studied here suggests not only that thetoxicity of aggregated species could be a general phenomenon, butalso that the pathogenicity of protein-deposition diseases could beprimarily related to the structural nature of the aggregates ratherthan to the specific sequences of the proteins from which they arise.We suggest that toxicity could primarily arise because on the surfaceof disordered aggregates there is likely to be a combinatorial displayof amino acids enabling these species to interact inappropriatelywith a wide range of cellular components. Such a conclusion furthersuggests that the differing clinical manifestations of amyloid for-mation, ranging from neuronal cell death to the accumulation oflarge quantities of proteinaceous material, could arise in large partbecause of variations in the nature and morphologies of the specificaggregates as they form in the different diseases, as well as on thesusceptibility of different cell types to the various aggregates. Suchvariations will occur as a consequence of the specific character of thedifferent proteins involved and their location, and also on thedifferent conditions under which aggregation takes place.

Implications for misfolding diseases and biological evolutionThe present findings, that early aggregates formed by a wider rangeof proteins than those known to be associated with neurologicaldiseases can be cytotoxic, provide new opportunities to define thenature of amyloid diseases and the mechanism of amyloid toxicity atthe molecular level. They also raise the possibility that traceamounts of aggregates of a variety of proteins might occur spon-taneously, particularly during ageing, and that such aggregatescould account for subtle impairments of cellular function in theabsence of an evident amyloid phenotype. It would thus be inter-esting to search for early protein aggregates in systemic andneurologic disorders not presently associated with amyloid for-mation. More generally, knowledge of the origin and nature ofaggregate pathogenicity is of crucial importance in efforts toidentify the correct targets for drug design in the search for effectivetherapeutic protocols.

The inherent toxicity in protein aggregates could also help us tounderstand fundamental aspects of cell biology. It suggests, forexample, that avoidance of aggregation could be more importantfor the proper functioning of biological organisms than waspreviously suspected if aggregates of proteins are often toxic ratherthan simply non-functional. In this case, in addition to increasingthe efficiency of folding and rescuing misfolded proteins afterbiosynthesis30, evolutionary developments to prevent aggregateformation, notably molecular chaperones, ubiquitination enzymesand proteasomes, are needed for the preservation of the long-termviability of living organisms. This latter idea is reinforced by recentfindings concerning the relationships between neurodegenerativediseases and failure of cellular defence mechanisms targettedtowards misfolded proteins and the existence, in both prokaryoticand eukaryotic cells, of a complex regulatory system of intracellularprotein degradation (see ref. 31 and references therein). Suchresults, together with findings that aggregate formation is linkedto the inheritance of specific traits in organisms such as yeasts32,

provides increasing evidence that the control of protein misfoldingand aggregation in addition to being of fundamental importancefor cell viability, has been a major driving force in biologicalevolution. A

MethodsProtein aggregate productionPI3-SH3 was expressed and purified as previously reported33. Granular aggregates wereformed incubating the protein for 1 h at 20 8C at a concentration of 10 mg ml21 in 50 mMacetate buffer, pH 5.5, containing 25% (v/v) TFE. Fibrillar aggregates were grown for 1month at a protein concentration of 10 mg ml21 in a water/HCl mixture, pH 2.0, at 37 8C.Conditions for HypF-N purification and aggregation have been described previously20.

Cell cultureNIH-3T3 cells (mouse fibroblasts, American Type Culture Collection) were routinelycultured in Dulbecco’s modified Eagle’s medium (Gibco BRL) containing 10.0% bovinecalf serum and 3.0 mM glutamine in a 5.0% CO2 humidified environment, at 37 8C. PC12cells (rat pheochromocytoma, American Type Culture Collection) were cultured in RPMImedium (Gibco BRL) supplemented with 10.0% horse serum, 5.0% fetal bovine serumand 3.0 mM glutamine in a 5.0% CO2 humidified atmosphere at 37 8C. 100 U ml21

penicillin and 100 mg ml21 streptomycin were added to both media. Cells were used for amaximum of 20 passages. NIH-3T3 or PC12 cells were plated at a density of 10,000 cellsper well on 96-well plates in 100 ml of fresh medium. After 24 h, the NIH-3T3 medium wasexchanged with 100 ml of DMEM, without phenol red, containing 10.0% bovine calfserum, and the PC12 medium was changed with 100 ml of RPMI, without phenol red,supplemented with 10.0% horse serum and 5.0% fetal bovine serum.

Incubation of cells in the presence of protein aggregatesAliquots of solutions containing native or aggregated proteins were centrifuged, driedunder N2 to remove TFE when necessary, dissolved in RPMI without phenol red andimmediately added to the cell media at 0.1–20 mM (PI3-SH3) or 0.04–20.0 mM (HypF-N)final concentrations. After 24 h incubation, 10 ml of a stock MTT solution in PBS wasadded to give a final concentration of 0.5 mg ml21 and incubated for a further 4 h. 100 ml ofcell lysis buffer (20.0% SDS, 50.0% N,N-dimethylformamide, pH 4.7) was added to eachwell and the samples were incubated overnight at 37 8C in an humidified incubator.Absorbance values of blue formazan were determined at 590 nm with an automatic platereader. Cell death was assessed by the trypan blue internalization test34. After a 24 hincubation with 48 h aged HypF-N in TFE or with the soluble domain, NIH-3T3 cells weretreated with trypan blue and survival was quantified by counting (three fields per well, twowells per condition, an average of 50 cells per field).

Electron microscopyTEM images were acquired using a JEM 1010 transmission electron microscope at 80 kVexcitation voltage. In each case, 3.0 ml of protein solution was placed on a formvar andcarbon-coated grid and blotted off after 3 min. The sample was then stained with 3 ml of2.0% uranyl acetate, dried and observed at a magnification of 12–30,000.

ThT staining60 ml aliquots of protein solution were mixed with 0.44 ml of 25 mM ThT in 25 mMphosphate buffer, pH 6.0, and the resulting fluorescence measured immediately aftermixing using a Shimadzu RF-5000 spectrofluorimeter at excitation and emissionwavelengths of 440 and 485 nm, respectively.

Received 2 January; accepted 11 February 2002.

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AcknowledgementsThe work was supported by grants from the Italian MIUR (PRIN “Folding e Misfolding diProteine) and the Italian Telethon Foundation. The research of C.M.D. is supported inpart by a Programme Grant from the Wellcome Trust. F.C. is supported by a fellowshipfrom the Italian Telethon Foundation.

Competing interests statement

The authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to M.S.

(e-mail: stefani@scibio.unifi.it) or C.M.D. (e-mail: cmd44@cam.ac.uk).

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