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• W2044448989 abstract "The intracellular free Ca2+ concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca2+ levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca2+-free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases. The intracellular free Ca2+ concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca2+ levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca2+-free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases. Approximately 20 different peptides or proteins, including the Aβ peptides, β2-microglobulin, transthyretin, lysozyme, α-synuclein, and the prion protein, are the main components of amyloid aggregates in vivo, each being associated with a specific disease such as Alzheimer's, Parkinson's, and prion diseases, type 2 diabetes, and systemic amyloidoses. In these pathologies it is likely that the impairment of cellular function is a direct consequence of the interaction of cellular components with protein aggregates that may be present, in some cases, at very low levels (1Thomas T. Thomas G. McLendon C. Sutton T. Mullan M. Nature. 1996; 380: 168-171Google Scholar, 2Pepys M.B. Wheaterall D.J. Ledingham J.G. Warrel D.A. Oxford Textbook of Medicine. 3rd Ed. Oxford University Press, Oxford1995: 1512-1524Google Scholar). In systemic non-neuropathic diseases, however, the accumulation in tissues and organs of large amounts of amyloid deposits may in itself be the primary origin of the clinical symptoms (2Pepys M.B. Wheaterall D.J. Ledingham J.G. Warrel D.A. Oxford Textbook of Medicine. 3rd Ed. Oxford University Press, Oxford1995: 1512-1524Google Scholar, 3Koo E.H. Lansbury P.T. Kelly J.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9989-9990Google Scholar). In some cases the proteins deposited are wild-type, as in sporadic amyloidoses, and in other cases they are modified by specific genetic mutations as in early onset familial diseases (4Kelly J. Curr. Opin. Struct. Biol. 1998; 8: 101-106Google Scholar, 5Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Google Scholar). Since 1998, an increasing number of natural or de novo designed sequences of proteins or peptides that are not associated with any known medical condition have been shown to aggregate in vitro into fibrils that are indistinguishable from those associated with the amyloid diseases (reviewed in Ref. 6Stefani M. Dobson C.M. J. Mol. Med. 2003; 81: 678-699Google Scholar). In the case of globular proteins, aggregation is found to require destabilizing conditions such that the proteins involved are at least partially unfolded but still able to form intramolecular interactions, notably those involving hydrogen bonding (7Chiti F. Webster P. Taddei N. Clark A. Stefani M. Ramponi G. Dobson C.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3590-3594Google Scholar). These and other findings indicate that amyloid aggregation is a generic property of the polypeptide chain, most probably linked to the common structure of the peptide backbone (7Chiti F. Webster P. Taddei N. Clark A. Stefani M. Ramponi G. Dobson C.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3590-3594Google Scholar, 8Dobson C.M. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2001; 356: 133-145Google Scholar, 9Dobson C.M. Nature. 2002; 418: 729-730Google Scholar). They also increase dramatically the number of proteins one can investigate to assess general features underlying protein misfolding and aggregation as well as the interaction of the aggregates with cells. Recently, evidence is beginning to emerge suggesting that the effects of aggregation on cellular function might be very similar for different proteins. In particular, aggregates of two proteins unrelated to disease, the SH3 1The abbreviations used are: SH, Src homology; HypF-N, HypF N-terminal domain; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; MOPS, 3-(N-morpholino)propanesulfonic acid; ROS, reactive oxygen species; CM-H2 DCFA, 2′,7′-dihydrodichlorofluorescein diacetate; AM, acetoxymethylester. 1The abbreviations used are: SH, Src homology; HypF-N, HypF N-terminal domain; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; MOPS, 3-(N-morpholino)propanesulfonic acid; ROS, reactive oxygen species; CM-H2 DCFA, 2′,7′-dihydrodichlorofluorescein diacetate; AM, acetoxymethylester. domain from the bovine phosphatidylinositol 3-kinase and the N-terminal domain of the Escherichia coli HypF (HypF-N) have been investigated from this perspective. Both of these proteins form amyloid fibrils under mild denaturing conditions (10Gujiarro J.I. Sunde M. Jones J.A. Campbell I.D. Dobson C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4224-4228Google Scholar, 11Chiti F. Bucciantini M. Capanni C. Taddei N. Dobson C.M. Stefani M. Protein Sci. 2001; 10: 2541-2547Google Scholar), and as observed for other amyloidogenic proteins, various prefibrillar aggregates are formed prior to the growth of mature fibrils, whose appearance often requires much longer periods of time (10Gujiarro J.I. Sunde M. Jones J.A. Campbell I.D. Dobson C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4224-4228Google Scholar, 11Chiti F. Bucciantini M. Capanni C. Taddei N. Dobson C.M. Stefani M. Protein Sci. 2001; 10: 2541-2547Google Scholar). Both proteins have been found to be toxic to cells only when added to the culture media in their prefibrillar state (12Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Google Scholar). The findings on the cytotoxicity of the proteins that are not associated with disease are closely similar to the conclusions drawn from studies on disease-associated proteins such as the Aβ peptides, α-synuclein, and transthyretin (1Thomas T. Thomas G. McLendon C. Sutton T. Mullan M. Nature. 1996; 380: 168-171Google Scholar, 13Conway K.A. Lee S.-J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Google Scholar, 14Bhatia R. Lin H. Lal R. FASEB J. 2000; 14: 1233-1243Google Scholar, 15Goldberg M.S. Lansbury P.T. Nat. Cell Biol. 2000; 2: E115-E119Google Scholar, 16Sousa M.M. Cardoso I. Fernandes R. Guimaraes A. Saraiva M.J. Am. J. Pathol. 2001; 159: 1993-2000Google Scholar, 17Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Google Scholar); these conclusions suggest that the cytotoxicity associated with aggregation may be determined by common features of specific types of aggregates rather than by specific amino acid sequences (12Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Google Scholar). The latter conclusion is also supported by recent findings providing evidence that the soluble prefibrillar aggregates of a range of different peptides and proteins are all recognized by polyclonal antibodies raised against a molecular mimic of soluble Aβ oligomers; the same antibodies fail to recognize the corresponding mature fibrillar aggregates, whereas they suppress the cytotoxicity of the prefibrillar aggregates (18Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Google Scholar). These findings indicate that the prefibrillar aggregates of proteins and peptides that form amyloid fibrils share common structural features that differ from those displayed both by their nonaggregated precursors and by the mature fibrils; they also argue against specific mechanisms of toxicity of the differing amyloid aggregates. Therefore, an increasing body of evidence supports the idea that the ability to form amyloid aggregates and the toxicity of these aggregates are generic properties of peptides and proteins. It also suggests that, in general, the most highly cytotoxic aggregates are the early prefibrillar assemblies or, possibly in some cases, the individual misfolded molecules (19Svanborg C. Agerstam H. Aronson A. Bjerkvig R. Duringer C. Fischer W. Gustafsson L. Hallgren O. Leijonhuvud I. Linse S. Mossberg A.-K. Nilsson H. Pettersson J. Svensson M. Adv. Cancer Res. 2003; 88: 1-29Google Scholar) rather than mature fibrils. To explore in depth the fundamental origins of the cytotoxicity of early aggregates of proteins that ultimately form amyloid fibrils, we have investigated the consequence of the exposure of murine fibroblasts (NIH-3T3 cells) to prefibrillar aggregates formed from the HypF-N domain and compared the results with findings reported previously with cells exposed to peptides and proteins that are specifically associated with amyloid diseases. This study has enabled us to explore whether the mechanisms by which protein aggregation results in cellular toxicity share a common basis or are protein-specific even in the case of proteins not associated with disease. In particular, we have examined those cellular characteristics found to be perturbed by aggregates of disease-associated proteins, notably the intracellular redox status and the levels of Ca2+ ions; we have also investigated the apoptotic or necrotic status of cells following prolonged exposure to the aggregates and explored the reversibility of cell damage occurring prior to cell death. We suggest, on the basis of our findings, that the toxicity of misfolded and aggregated states of different peptides and proteins proceeds through the modification of the same specific biochemical properties in the exposed cells and that such a modification depends on the type of aggregate rather than on the specific amino acid sequence of the aggregated protein. It can therefore be predicted that the cascade of biochemical modifications triggered by the exposure of cells to any aggregated polypeptide chain ultimately leading to cell death, at least in most cases, starts with the alteration of the same cellular parameters. Reagents—Dichlorodihydrofluorescein diacetate (CM-H2 DCFA), Fura2-AM, Fluo-3-AM, Texas Red STP ester sodium salt, and wheat germ agglutinin-conjugated fluorescein were from Molecular Probes (Eugene, OR). The caspase 3 assay, cell culture media, materials for microscopy analysis, and other reagents were obtained from Sigma-Aldrich. NIH-3T3 murine fibroblasts were routinely cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% bovine calf serum, unless otherwise stated and 3.0 mm glutamine in a 5% CO2 humidified environment at 37 °C. 100.0 units/ml penicillin and 100.0 μg/ml streptomycin were added to the media. The cells were used for a maximum of 20 passages. HypF-N was purified as described previously (11Chiti F. Bucciantini M. Capanni C. Taddei N. Dobson C.M. Stefani M. Protein Sci. 2001; 10: 2541-2547Google Scholar). Rabbit anti-HypF-N polyclonal antibodies were provided by Primm S.r.l. (Milan, Italy); Alexa-488-conjugated anti-rabbit IgG secondary antibodies were from Molecular Probes. Formation and Labeling of Prefibrillar Aggregates—Prefibrillar aggregates of HypF-N were obtained by incubating the protein for 48 h at room temperature at a concentration of 0.3 mg ml–1 in 30% (v/v) trifluoroethanol, 50 mm sodium acetate, pH 5.5. The solution was centrifuged, and the resulting pellet was dried under N2 to remove the remaining solvent, dissolved in RPMI serum-free medium, and immediately added to the culture medium of cells grown on coverslips to a final protein concentration of 20 μm. The presence of HypF-N aggregates inside cells was detected by labeling preformed aggregates with Texas Red as follows. To 1.0 mg of the protein aggregates dissolved in 0.1 ml of 0.1 m sodium bicarbonate buffer, pH 8.5, 10 μl of a 10 mg/ml Texas Red solution in Me2SO was added slowly. After 1 h at room temperature with continuous stirring, the reaction was stopped by adding 10 μl of 1.5 m hydroxylamine, pH 8.5. The cells were then counterstained with fluorescein-conjugated wheat germ agglutinin (50 μg/ml) for 15 min to detect plasma membrane profiles. Coverslips containing the cells and the labeled prefibrillar aggregates were then mounted on the stage of a confocal Bio-Rad MCR 1024 ES scanning microscope (Bio-Rad) equipped with a krypton/argon laser source (15 mW) for fluorescence measurements. Observations were performed using a Nikon Plan Apo 60× oil immersion objective. Texas Red fluorescence was revealed at 568-nm excitation, and fluorescein was revealed at 488-nm excitation. A series of optical sections (512 × 512 pixels) 1.0 μm in thickness was taken through the cells at intervals of 0.8 μm. 20 optical sections for each examined sample were then projected as single composite image by superimposition. In the indirect immunofluorescence experiments, the cells were plated on glass coverslips and incubated with the prefibrillar aggregates. Then the cells were fixed in 2.0% buffered paraformaldehyde for 10 min at room temperature and blocked with a 0.5% bovine serum albumin and 3.0% glycerol solution in PBS. After washing, the coverslips were incubated with rabbit polyclonal anti-HypF-N antibodies diluted 1:100 in PBS with bovine serum albumin for 60 min. The immunoreaction was revealed with Alexa-488-conjugated anti-rabbit secondary antibodies (Molecular Probes) diluted 1:200. Negative controls were obtained by substituting blocking solution for the primary antibody. Counterstaining for the nuclei was performed with propidium iodide. The fluorescence was analyzed by confocal laser microscopy (Bio-Rad) using two emission lines at 488 and 568 nm for Alexa-488 and propidium iodide excitation, respectively. Assay of Cellular Toxicity—Aggregate cytotoxicity was assessed by the MTT reduction inhibition assay (20Abe K. Kimura H. J. Neurochem. 1996; 67: 2074-2078Google Scholar). NIH-3T3 cells were plated on to 96-well plates at a density of 10,000 cells/well in 100 μl of fresh medium. After 24 h, the culture medium was exchanged with 100 μl of RPMI serum-free medium without red phenol. The cells were exposed to the HypF-N aggregates for differing lengths of time as previously reported (12Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Google Scholar). 10.0 μl of a stock MTT solution in PBS was then added to the cells to a final concentration of 0.5 mg/ml, and the incubation was continued for a further 2 h. At this time, cell lysis buffer (100 μl/well; 20% SDS, 50% N,N-dimethylformamide, pH 4.7) was added, and the samples were incubated overnight at 37 °C in a humidified incubator. The absorbance of blue formazan was measured at 570 nm using an automatic plate reader as described previously (12Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Google Scholar). Control experiments were performed by exposing cells to solutions of the nonaggregated, monomeric protein (final concentration, 20.0 μm) for the same lengths of time. Reversibility of Cell Damage—Reversibility of cell impairment was assessed by performing the MTT assay on cells cultured in serum-free media to avoid cell duplication; the cells were incubated with a serum-free medium containing 20.0 μm of aggregated protein for different lengths of time, washed, and cultured in serum-free medium for 24 h. As a control, the cells were exposed to a serum-free medium containing the same amount of soluble HypF-N. To assess the role of proteasomal involvement, the cells were treated with 10.0 μm lactacystin for varying lengths of time before incubation with a serum-free medium containing 20.0 μm of protein aggregates; at the end of the incubation period, the cells were cultured for 24 h in fresh, serum-free medium and then subjected to the MTT assay. The controls were prepared by incubating cells with lactacystin and then exposing them to 20.0 μm soluble HypF-N for the same lengths of time. Measurement of the Production of Intracellular Reactive Oxygen Species—Intracellular ROS assays were performed using the ROS-sensitive fluorescent probe CM-H2 DCFA. NIH-3T3 cells were seeded in 6-well plates at 5 × 104 cells/0.8 ml/well. After 24 h, the cells were incubated in the presence of 20.0 μm aggregated protein for differing lengths of time. At the end of the incubation, the cells were washed with PBS and loaded with 5.0 μm CM-H2 DCFA in the culture medium for 15 min at 37 °C, washed with PBS, and then lysed with RIPA buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 100 mm NaF, 2.0 mm EGTA, 1.0 mm sodium vanadate, 1% Triton, 10.0 μg/ml aprotinin, 10.0 μg/ml leupeptin). ROS levels were detected in the samples by measuring the fluorescence of the oxidized CM-H2 DCFA with a PerkinElmer Life Sciences 55 spectrofluorimeter (Wellesley, MA), with excitation and emission wavelengths of 488 and 520 nm, respectively). CM-H2 DCFA fluorescence into intact cells was also detected using a confocal Bio-Rad MCR 1024 ES scanning microscope equipped with a krypton/argon laser source (15 mW). A series of optical sections (512 × 512 pixels) was taken through the depth of the cells with a thickness of 1.0 μm at intervals of 0.8 μm. Twenty optical sections for each examined sample were then projected as a single composite image by superimposition. The time course analysis of ROS production was performed using the software Time Course Kinetic (Bio-Rad). Measurements of Intracellular Free Ca2+ Levels—The levels of free Ca2+ present in the cytosol were measured using the fluorescent probes Fura2-AM or Fluo-3-AM (21Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Google Scholar). Subconfluent plates were incubated for differing lengths of time in the presence of a solution containing 20.0 μm of protein aggregates. The cells were washed with PBS and incubated in Dulbecco's modified Eagle's medium supplemented with 10.0% fetal calf serum containing 10.0 μm Fura2-AM. To increase the effectiveness of the probe, Fura2-AM stock solutions (10 mm in dry Me2SO) were mixed in a 1:1 ratio with 20% pluronic acid F127 prior to addition to the cell medium. Incubation was carried out at 37 °C for 1 h, and then the extracellular dye was removed by washing with a large excess of 10 mm MOPS buffer, pH 7.0, containing 115 mm KCl, 20 mm NaCl, 1.0 mm EGTA, and 1.0 mm MgCl2. The cells were resuspended in 50 μl of the same buffer, counted, and then diluted to a density of 1.0 × 106 cells/ml in a spectrofluorimetric cuvette thermostatted at 25 °C. Intracellular Ca2+ concentrations were then calculated using standard protocols (21Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Google Scholar). In a separate set of experiments intracellular Ca2+ was imaged in intact cells using the Ca2+-sensitive probe Fluo-3 and the laser confocal microscope used for intracellular ROS imaging. The cells were cultured on collagen IV-coated glass coverslips, and dye loading was performed by incubating cells with 5.0 μm Fluo-3 for 20 min at 37 °C in the culture medium. Then the coverslips were mounted in a chamber and placed on the stage of the confocal microscope. Fluo-3 fluorescence was monitored at a wavelength of 488 nm by collecting the emitted fluorescence with a Nikon Plan Apo 60× oil immersion objective through a 510-nm-long wave pass filter. The time course analysis of intracellular calcium was performed using the software Time Course Kinetic (Bio-Rad). Assays for Cell Apoptosis—The extent to which the apoptotic pathway had been triggered in 80% confluent cells exposed for 48 h to a 20 μm final concentration of HypF-N aggregates was determined by measuring caspase-3 activation using a colorimetric 96-well plate assay system (Sigma) as specified by the provider. An important question in the investigation of protein aggregate toxicity is to assess whether the effects of aggregates to cells follow aggregate internalization. We first investigated whether the prefibrillar aggregates of HypF-N added to the cell culture medium appeared in the cytoplasm, by labeling the aggregates with the fluorescent probe Texas Red. Fig. 1 shows confocal microscopy images at different focal lengths of cells exposed for 3 h to the labeled HypF-N aggregates; similar images were seen after 1 h of exposure (not shown). The aggregated species can be seen to have accumulated in the vicinity of the plasma membrane (Fig. 1A) and also to be present in the intracellular focal plane (Fig. 1B, arrows). The ingress of the aggregates into the cells does not result simply from the enhanced hydrophobicity of the aggregate-Texas Red complex, because similar data were obtained using unlabeled aggregates whose presence inside cells was detected by immunofluorescence using polyclonal anti-HypF-N antibodies (Fig. 1C). This result shows that early aggregates of HypF-N added to the cell culture medium are able to be internalized, although the mechanism of aggregate translocation inside cells requires further investigation. The biochemical parameters we investigated in the cells exposed to the prefibrillar aggregates were the redox status and the intracellular free Ca2+. Fig. 2 shows the results of the experiments aimed at determining the levels of ROS that are generated within cells following exposure for 3 h to 20 μm HypF-N in both soluble and aggregated forms. A significant increase in ROS, concomitant with a decrease in cell viability as detected by the MTT assay, was found (Fig. 2A). ROS increase and cytotoxicity, however, were not present in cells incubated, prior to exposure to the aggregates, in the presence of 0.10 mm α-tocopherol (vitamin E), a lipid-soluble antioxidant that is very effective in suppressing membrane lipid peroxidation (Fig. 2A). Similar results were obtained using propylgallate or promethazine as reducing agents (data not shown). The ROS levels after exposure to HypF-N aggregates were also determined by monitoring cells with confocal microscopy in the presence of the fluorescent probe CM-H2 DCFA. As in the experiments performed using cell lysates, a considerable increase in ROS levels was found in living cells exposed to the aggregates, whereas no ROS increase was observed when the same cells were treated with 0.10 mm α-tocopherol (Fig. 2B). These findings show that the HypF-N prefibrillar aggregates modify the redox status of cells in a manner closely similar to that previously reported for peptides and proteins that are associated with protein deposition diseases (see “Discussion”). To assess whether the HypF-N aggregates induced an increase of intracellular Ca2+ levels, cell media were supplemented with a 1:1 mixture of 10.0 μm Fura2-AM, 20% pluronic acid F127 (see “Experimental Procedures”). Fig. 3A shows that intracellular Ca2+ levels rise by a factor of about 2 in cells exposed for 3 h to a 20 μm solution of aggregated protein compared with control experiments where cells were incubated in the presence of the same quantity of soluble HypF-N. This increase, along with a concomitant decrease in cell viability, as determined by the MTT test, was abolished when the cells were cultured in a Ca2+-free medium (Fig. 3A). The intracellular Ca2+ levels after exposure to the HypF-N aggregates were also monitored in vivo by confocal microscopy (see “Experimental Procedures”). A considerable increase in Ca2+ was found in cells exposed to the aggregates, whereas no increase was observed when cells were cultured in a Ca2+-free medium (Fig. 3B). These data reveal that intracellular Ca2+ levels are modified in the cells exposed to the HypF-N aggregates in a manner similar to that reported previously for cells incubated in the presence of disease-associated peptides and proteins including the Aβ peptide, human islet amylin, the amyloidogenic prion protein fragment, and Cu,Zn-SOD (see “Discussion”). Intracellular Ca2+ and ROS levels were also monitored by confocal microscopy in cells incubated in the presence of α-tocopherol prior to exposure to the HypF-N aggregates and in cells cultured in Ca2+-free medium during the time they were exposed to the aggregates, respectively. Fig. 4 shows that the ROS levels are high following exposure to aggregates even in the absence of Ca2+ in the medium. By contrast, free Ca2+ levels in cells pretreated with α-tocopherol before exposure to HypF-N aggregates were considerably lower than in cells not treated with α-tocopherol and the same as in control cells exposed to the protein in its soluble form. The substantial increase in ROS caused by the HypF-N aggregates even in the absence of Ca2+ in the medium indicates that oxidative stress is, at least in part, independent of the rise of the intracellular Ca2+ levels. This conclusion is supported by the observed time courses of both Ca2+ and ROS increases, where the ROS increase is found to precede the Ca2+ rise during the first 20 min of exposure to the aggregates (Fig. 5). However, both events occur rapidly compared with other changes in cellular functions such as apoptosis or necrosis, being almost complete after 60 min of exposure. The finding that the viability of cells cultured in Ca2+-free medium is unaffected by exposure to the HypF-N aggregates, although displaying enhanced ROS production, indicates that, at least under our conditions, oxidative stress does not by itself generate cytotoxicity. We have also investigated the degree of reversibility of the rise of both intracellular ROS and Ca2+ levels, along with the associated cell damage. The effect of preincubation of cells in the presence of lactacystin (10.0 μg/ml), a potent proteasome inhibitor, before exposure to the aggregates was also examined. Cells were cultured in serum-free medium to avoid cell division following transfer in aggregate-free fresh medium. Fig. 6 shows that cell damage appears to be almost completely reversible after exposure to the aggregates for relatively short lengths of time (less than 5 h) when the cells almost completely recover the ability to reduce MTT. For longer times of exposure (until 15 h) cells recovered viability only partially. Moreover, the cells stressed upon pretreatment with lactacystin followed by exposure to the aggregates were more susceptible to damage than were untreated cells; in this case, the reversal of the toxic effects was dependent upon the length of the time of preincubation with lactacystin. After 15 h of preincubation, the cell damage was not reversed when the cells were moved to fresh media, whereas for 5 and 3 h of preincubation, reversal was partial or complete, respectively, indicating a different degree of impairment of the mechanisms of clearance of the aggregates. It has previously been reported that, after prolonged exposure to the HypF-N aggregates, cells undergo death, as shown by the trypan blue exclusion assay (12Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Google Scholar). We have not investigated in detail the features of cell death under our conditions; however, we found a necrotic rather than an apoptotic condition in our cells exposed for 24 h to the aggregates (data not shown). Nevertheless, a progressive caspase-3 activation was initially present, reaching a maximum (around 250%) after 6–7 h of exposure to the aggregates, indicative of an apoptotic response (data not shown). Although preliminary, these data suggest that, in our exposed cells, the apoptotic pathway is initially triggered but is not sustained and is followed by necrotic death. A deeper investigation of the biochemical features of cell death following exposure to the toxic aggregates is presently underway. It is now well established that the aggregation process of peptides and proteins leadi" @default.
• W2044448989 created "2016-06-24" @default.
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• W2044448989 date "2004-07-01" @default.
• W2044448989 modified "2023-10-14" @default.
• W2044448989 title "Prefibrillar Amyloid Protein Aggregates Share Common Features of Cytotoxicity" @default.
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