Calcium Dysregulation and Membrane Disruption as a Ubiquitous Neurotoxic Mechanism of Soluble Amyloid Oligomers*♦

Increasing evidence suggests that amyloid peptides associated with a variety of degenerative diseases induce neurotoxicity in their intermediate oligomeric state, rather than as monomers or fibrils. To test this hypothesis and investigate the possible involvement of Ca2+ signaling disruptions in amyloid-induced cytotoxicity, we made homogeneous preparations of disease-related amyloids (Aβ, prion, islet amyloid polypeptide, polyglutamine, and lysozyme) in various aggregation states and tested their actions on fluo-3-loaded SH-SY5Y cells. Application of oligomeric forms of all amyloids tested (0.6–6 μgml–1) rapidly (∼5 s) elevated intracellular Ca2+, whereas equivalent amounts of monomers and fibrils did not. Ca2+ signals evoked by Aβ42 oligomers persisted after depletion of intracellular Ca2+ stores, and small signals remained in Ca2+-free medium, indicating contributions from both extracellular and intracellular Ca2+ sources. The increased membrane permeability to Ca2+ cannot be attributed to activation of endogenous Ca2+ channels, because responses were unaffected by the potent Ca2+-channel blocker cobalt (20 μm). Instead, observations that Aβ42 and other oligomers caused rapid cellular leakage of anionic fluorescent dyes point to a generalized increase in membrane permeability. The resulting unregulated flux of ions and molecules may provide a common mechanism for oligomer-mediated toxicity in many amyloidogenic diseases, with dysregulation of Ca2+ ions playing a crucial role because of their strong trans-membrane concentration gradient and involvement in cell dysfunction and death. Increasing evidence suggests that amyloid peptides associated with a variety of degenerative diseases induce neurotoxicity in their intermediate oligomeric state, rather than as monomers or fibrils. To test this hypothesis and investigate the possible involvement of Ca2+ signaling disruptions in amyloid-induced cytotoxicity, we made homogeneous preparations of disease-related amyloids (Aβ, prion, islet amyloid polypeptide, polyglutamine, and lysozyme) in various aggregation states and tested their actions on fluo-3-loaded SH-SY5Y cells. Application of oligomeric forms of all amyloids tested (0.6–6 μgml–1) rapidly (∼5 s) elevated intracellular Ca2+, whereas equivalent amounts of monomers and fibrils did not. Ca2+ signals evoked by Aβ42 oligomers persisted after depletion of intracellular Ca2+ stores, and small signals remained in Ca2+-free medium, indicating contributions from both extracellular and intracellular Ca2+ sources. The increased membrane permeability to Ca2+ cannot be attributed to activation of endogenous Ca2+ channels, because responses were unaffected by the potent Ca2+-channel blocker cobalt (20 μm). Instead, observations that Aβ42 and other oligomers caused rapid cellular leakage of anionic fluorescent dyes point to a generalized increase in membrane permeability. The resulting unregulated flux of ions and molecules may provide a common mechanism for oligomer-mediated toxicity in many amyloidogenic diseases, with dysregulation of Ca2+ ions playing a crucial role because of their strong trans-membrane concentration gradient and involvement in cell dysfunction and death. Alzheimer disease (AD) 1The abbreviations used are: AD, Alzheimer disease; IAPP, islet amyloid polypeptide. 1The abbreviations used are: AD, Alzheimer disease; IAPP, islet amyloid polypeptide. is characterized by the appearance in the brain of plaques, containing extracellular deposits of amyloid β-peptide (Aβ) that result from altered proteolytic processing of amyloid precursor protein, together with intracellular neurofibrillary tangles containing misfolded tau (1Mattson M.P. 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Moreover, soluble oligomeric forms of several amyloids (including islet amyloid polypeptide (IAPP), α-synuclein, prion, and polyglutamine) are, respectively, implicated as the primary toxic species in type 2 diabetes mellitus, Parkinson disease, spongiform encephalopathies, and Huntington disease (6Lashuel H.A. Hartley D.M. Balakhaneh D. Aggarwal A. Teichberg S. Callaway D.J. J. Biol. Chem. 2002; 277: 42881-42890Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 15Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3405) Google Scholar, 18Caughey B. Lansbury P.T. Annu. Rev. Neurosci. 2003; 26: 267-298Crossref PubMed Scopus (1433) Google Scholar). This toxicity of amyloid oligomers appears to be intrinsically related to their aggregation state, because oligomeric forms of proteins and peptides that are not disease-related are equally toxic (14Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2132) Google Scholar, 19Bucciantini M. Calloni G. Chiti F. Formigli L. Nosi D. Dobson C.M. Stefani M. J. Biol. Chem. 2004; 279: 31374-31382Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Taken together, these findings support the notion of “conformational disease” (20Carrell R.W. Lomas D.A. Lancet. 1997; 350: 134-138Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar), whereby a common structural motif formed by otherwise unrelated amyloid oligomers leads to a common mechanism of pathogenesis (14Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2132) Google Scholar, 15Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3405) Google Scholar). However, it remains to be established precisely which aggregation state is primarily responsible for the neurotoxicity, and difficulties in preparing highly homogeneous and stable populations of monomers, oligomeric intermediates, and fibrils may account for observations of highly variable actions among different cells (21Kawahara M. Kuroda Y. Arispe N. Rojas E. J. Biol. Chem. 2000; 275: 14077-14083Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar).Questions also remain regarding the mechanism of amyloid toxicity. Growing evidence points to a disruption of intracellular Ca2+ homeostasis in AD and other amyloidogenic diseases (1Mattson M.P. Nature. 2004; 430: 631-639Crossref PubMed Scopus (2429) Google Scholar, 21Kawahara M. Kuroda Y. Arispe N. Rojas E. J. Biol. Chem. 2000; 275: 14077-14083Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 22Blanchard B.J. Chen A. Rozeboom L.M. Stafford K.A. Weigele P. Ingram V.M. Proc. Natl. Acad. Sci. U. S. 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Prefibrillar amyloid aggregates have been shown to elevate cytosolic Ca2+ in neurons (19Bucciantini M. Calloni G. Chiti F. Formigli L. Nosi D. Dobson C.M. Stefani M. J. Biol. Chem. 2004; 279: 31374-31382Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 21Kawahara M. Kuroda Y. Arispe N. Rojas E. J. Biol. Chem. 2000; 275: 14077-14083Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), but such actions have been proposed to be secondary to the generation of reactive oxygen species (1Mattson M.P. Nature. 2004; 430: 631-639Crossref PubMed Scopus (2429) Google Scholar, 29Schubert D. Behl C. Lesley R. Brack A. Dargusch R. Sagara Y. Kimura H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1989-1993Crossref PubMed Scopus (363) Google Scholar), to arise from activation of cell surface receptors coupled to Ca2+ influx (22Blanchard B.J. Chen A. Rozeboom L.M. Stafford K.A. Weigele P. Ingram V.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14326-14332Crossref PubMed Scopus (136) Google Scholar, 30Guo Q. Furukawa K. Sopher B.L. Pham D.G. Xie J. Robinson N. Martin G.M. Mattson M.P. Neuroreport. 1996; 8: 379-383Crossref PubMed Scopus (299) Google Scholar, 31Mattson M.P. Chan S.L. Nat. Cell Biol. 2003; 5: 1041-1043Crossref PubMed Scopus (351) Google Scholar), or to result from Ca2+ influx across the plasma membrane as a result of either cation-selective channels formed by Aβ itself (21Kawahara M. Kuroda Y. Arispe N. Rojas E. J. Biol. Chem. 2000; 275: 14077-14083Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 33Kagan B.L. Hirakura Y. Azimov R. Azimova R. Lin M.C. Peptides. 2002; 23: 1311-1315Crossref PubMed Scopus (203) Google Scholar) or through a general disruption of lipid integrity (34Kayed R. Sokolov Y. Edmonds B. MacIntire T.M. Milton S.C. Hall J.E. Glabe C.G. J. Biol. Chem. 2004; 279: 46363-46366Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar).To address these issues we prepared pure samples of monomeric, oligomeric, and fibrillar forms of several disease-related amyloids and examined their respective actions on cytosolic Ca2+ levels in SH-SY5Y cells. We show that amyloid oligomers consistently produce rapid and dramatic elevations in [Ca2+], whereas equivalent concentrations of monomers or fibrils do not. The action of amyloid oligomers appears to involve a channel-independent disruption of the integrity of both plasma and intracellular membranes, and we propose that this may be a neurotoxic mechanism common to many amyloidogenic diseases.EXPERIMENTAL PROCEDURESMaterials—Fluorescent dyes (fluo-3AM, calcein) and thapsigargin were from Molecular Probes (Eugene, OR). Ionomycin, cell culture media, and other reagents were obtained from Sigma-Aldrich. Peptides were synthesized as described previously (35Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Abstract Full Text PDF PubMed Google Scholar). Lyophilized peptides and proteins were resuspended in 50% acetonitrile in water and re-lyophilized.Preparation of Homogeneous Populations of Amyloid Peptide Monomers, Oligomers, and Fibrils—Soluble monomer and oligomers were prepared by dissolving 1.0 mg of peptide in 400 μl of hexafluoroisopropanol for 10–20 min at room temperature. 100 μl of the resulting seedless solution was added to 900 μl double-distilled water in a siliconized Eppendorf tube. After 10–20-min incubation at room temperature, the samples were centrifuged for 15 min at 14,000 × g, and the supernatant fraction (pH 2.8–3.5) was transferred to a new siliconized tube and subjected to a gentle stream of N2 for 5–10 min to evaporate the hexafluoroisopropanol. The monomer solutions were used immediately after evaporation of hexafluoroisopropanol. For soluble oligomers, the samples were then stirred at 500 rpm using a Teflon-coated microstir bar for 24–48 h at 22 °C. Aliquots (10 μl) were taken at 6–12-h intervals for observation by electron microscopy and size exclusion chromatography. For prion 106–126, the samples were heated at 65 °C for 30 min before stirring. Poly(Q) was heated for 2 h at 37 °C.Fibrils were prepared as described above for oligomers, except they were stirred at room temperature for 6–9 days and sodium azide was added (0.02%). The final peptide concentration was 0.3 mg/ml. Fibril formation was monitored by thioflavin T fluorescence and UV light scattering. Once fibril formation was complete, the solutions were centrifuged at 14,000 × g for 20 min, the fibril pellet was washed three times with double-distilled water and then resuspended in the desired buffer. The morphology was verified by negative stain electron microscopy as reported previously (34Kayed R. Sokolov Y. Edmonds B. MacIntire T.M. Milton S.C. Hall J.E. Glabe C.G. J. Biol. Chem. 2004; 279: 46363-46366Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar).Cell Culture and Dye Loading—SH-SY5Y human neuroblastoma cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, glutamine (4 mm), penicillin (200 units/ml), streptomycin (200 μg/ml), and sodium pyruvate (1 μm). Cells were maintained at 37 °C in 5% CO2, and the medium was replaced every 2 days. Cells (∼10,000) were plated in 35-mm glass-bottom culture dishes (MatTek Corp.) and grown overnight. Loading with the Ca2+ indicator fluo-3 was accomplished by incubating with fluo-3-AM (7 μm in Hanks' balanced salt solution) for 30 min at room temperature, washing 3 times with Hanks' balanced salt solution and maintaining at 37 °C for 20 min to ensure complete hydrolysis. A similar loading protocol was utilized to load cells with calcein by incubating with 7 μm calcein-AM.Fluorescence Imaging—The imaging system consisted of an inverted microscope (Olympus IX 71) equipped with a Leitz 16X objective. Fluorescence excitation was by a 488 nm argon ion laser, and emitted fluorescence (λ > 510 nm) was imaged by a cooled CCD camera (Cascade 650, Roper Scientific). Time-lapse images (1 frame s–1) were captured using the MetaMorph software package (Universal Imaging, Westchester, PA), and fluorescence intensities were measured from regions of interest centered on individual cells. Signals are expressed as a pseudo ratio (ΔF/F) of the change in fluorescence (ΔF) divided by the resting fluorescence before treatment (F). A small proportion (9%) of cells failed to load with fluo-3, having low initial fluorescence and failing to respond to ionomycin. These were excluded from analysis.Amyloids were applied by pipetting a fixed aliquot (70 μl) of a diluted stock solution into the recording chamber (1-ml volume) directly above the microscope objective. To estimate the resulting concentration experienced by the cells under observation we pipetted the same volume of a fluorescent dye (calcein) into the chamber and measured the resulting fluorescence in the vicinity of the cells relative to that of the initial, undiluted solution of dye. This calibration yielded a dilution factor of about 5, which was assumed in calculating the effective concentrations of amyloids.To avoid complications from differences in molecular weights between monomers, oligomers and fibrils, we express these concentrations in units of μgml–1. As a rough guide, a concentration of 0.6 μg/ml Aβ42 corresponds to 200 nm monomer and ∼7 nm oligomer and 70 pm fibrils.RESULTSAβ42 Oligomers, but Not Monomers or Fibrils, Increase Intracellular Free Ca2+—Homogeneous populations of monomeric, oligomeric, and fibrillar Aβ were prepared as described above and characterized by size exclusion chromatography and electron microscopy. The oligomeric preparation had an approximate molecular mass of 90 kDa, contained very little material of lower molecular mass, and was comprised of spherical vesicles with diameters of 2–5 nm (Fig. 1). The monomeric preparation contained no detectable oligomeric aggregates as analyzed by size exclusion chromatography (Fig. 1). The morphology of the fibrillar preparations was as published previously (34Kayed R. Sokolov Y. Edmonds B. MacIntire T.M. Milton S.C. Hall J.E. Glabe C.G. J. Biol. Chem. 2004; 279: 46363-46366Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar).The actions of homogeneous monomeric, oligomeric, and fibrillar preparations of soluble Aβ42 amyloid were examined by adding aliquots of the samples to fluo-3-loaded SH-SY5Y cells (Fig. 2). Fig. 2A illustrates images and corresponding Ca2+-dependent fluorescence measurements in a representative cell. Applications of monomers or fibrils at final concentrations of 6 μg/ml evoked no detectable change in fluorescence, whereas subsequent application of the same amount of oligomer evoked large and rapid (∼5 s) increases in Ca2+-dependent fluorescence. Similar results were obtained in >200 cells examined. For example, Fig. 2B shows mean fluorescence signals from 22 cells, where Aβ42 oligomers evoked a fluo-3 signal with a peak increase of ΔF/F = 3.3 ± 0.5 (S.E.).Fig. 2Aβ42 oligomers, but not monomers or fibrils, elevate intracellular free [Ca2+].A, time course of Ca2+-dependent fluorescence recorded from a single fluo-3-loaded SH-SY5Y cell in response to sequential application of monomers, fibrils, and oligomers of Aβ42 (each at a final concentration of 6 μg/ml). The inset images of the cell were captured at the times indicated during the trace and are depicted on a pseudo color scale with “warmer” colors on a rainbow scale corresponding to higher fluorescence. B, trace shows the average responses from 22 cells, evoked using the same protocol. Error bars indicate ± 1 S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The fact that monomeric and fibrillar samples were inactive rules out the possibility that the increase in Ca2+ was due to trace contaminants in the solvent, because the solvent remained the same over the time of incubation for the conversion of monomer to oligomer and then ultimately to fibrils. Solvent controls in the absence of peptide had no effect on Ca2+ levels (data not shown).Actions of Other Disease-related Amyloids—To explore the possibility of a common neurotoxic mechanism among amyloidogenic diseases, we selected five disease-related amyloid peptides (Aβ42, prion, IAPP, polyglutamine, and lysozyme) and tested the effects of purified monomers, oligomers and fibrils of each of these amyloids on cytosolic Ca2+. Fig. 3A illustrates representative fluorescence records obtained with IAPP at a concentration of 6 μg/ml. Applications of IAPP monomers and fibrils evoked no detectable Ca2+ signals, even though the same cells showed large increases in fluorescence when subsequently challenged with the Ca2+ ionophore ionomycin (Fig. 3A, traces a and b). In marked contrast, equivalent amounts of purified IAPP oligomers produced a large fluorescence signal (Fig. 3A, trace c), closely resembling the response evoked by Aβ42 oligomers.Fig. 3Ca2+ elevations induced by other amyloids depend upon their aggregation state.A, fluorescence records illustrating typical responses to applications of 6 μg/ml of Aβ42 monomer (trace a), fibrils (trace b), and oligomers (trace c). Each trace was obtained from a different cell. No responses were observed to monomers or fibrils, even though the same cells gave large signals when subsequently challenged with ionomycin (6 μm). B, pooled data showing mean fluorescence signals (+S.E.) from 40 to 200 cells evoked by Aβ42, prion, IAPP, poly(Q), and lysozyme, each at a final concentration of 6 μg/ml. Measurements were obtained from records like those in A. For each amyloid, the histogram bars show responses evoked by monomers, fibrils, and oligomers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mean results obtained with all five amyloids are summarized in Fig. 3B. In each case the oligomeric forms evoked large increases in intracellular [Ca2+] (respective peak ΔF/F values Aβ42 = 3.3 ± 0.08; prion = 2.18 ± 0.1; IAPP = 2.45 ± 0.19; polyglutamine = 3.58 ± 0.07; lysozyme = 2.77 ± 0.1), whereas monomers and fibrils failed to produce any appreciable signal. Among a total of 1320 cells analyzed, 1280 showed Ca2+ responses to oligomers, whereas we never observed appreciable (ΔF/F > 0.4) fluorescence increases with monomers or fibrils.Concentration Dependence of Oligomer Action—We next examined the concentration dependence of the Ca2+ signals on oligomer concentration, using IAPP and Aβ42 as representative amyloids. The protocol is illustrated in Fig. 4A, and involved sequential additions of oligomer to the imaging chamber, resulting in stepwise increases in concentration. Concentrations of IAPP oligomer as low as 0.6 μg/ml (corresponding to 7.5 nm assuming a molecular mass of 80 kDa) already evoked a detectable fluorescence increase, and 12 μg/ml (about 150 nm) produced a maximal response, as assessed by the failure of ionomycin to cause any further increase in fluorescence (data not shown). Fig. 4B shows mean measurements of relative fluorescence signals evoked by increasing concentrations of Aβ42 and IAPP oligomers. Both amyloids showed similar concentration-response relationships, with half-maximal responses at respective concentrations of 2.7 and 3.5 μg/ml. These values may, however, underestimate the true EC50 of the oligomers if the indicator dye were saturated at higher concentrations.Fig. 4Dose dependence of Aβ42 and IAPP oligomers.A, trace shows Ca2+-dependent fluorescence signals averaged from 26 cells in response to successive additions of IAPP oligomers, resulting in stepwise concentration increases to final levels of 0.6, 2.4, 6, and 12 μg/ml. The inset images show pseudo color representations of a single cell, captured at the times indicated by the trace. B, mean peak fluorescence signals evoked by different concentrations of Aβ (red bars) and IAPP (blue bars) oligomers. Data are from 78 cells for IAPP and 85 for Aβ42 and are expressed as a percentage of the maximal response evoked by 12 μg/ml oligomer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Elevation of Cytosolic Free Ca2+from Aβ42 Oligomers Involves Both Intra- and Extracellular Sources—To discriminate whether the increase in cytosolic Ca2+ concentration evoked by amyloid oligomers arises from influx of extracellular Ca2+ across the plasma membrane or from Ca2+ liberated from intracellular stores, we reduced the free extracellular Ca2+ concentration to very low levels by bathing cells for 20 min in a Ca2+-free medium that included 5 mm Mg2+ and 10 mm EGTA. Application of Aβ42 oligomers (6 μg/ml) then evoked only a small, slowly rising fluorescence signal, which reached a peak amplitude of about 30% of that in Ca2+-containing medium (Fig. 5A). Thus, the large, immediate Ca2+ increase induced by oligomers appears to result from influx of extracellular Ca2+, whereas the remaining small, slow component may arise from intracellular Ca2+ liberation.Fig. 5Aβ42 oligomer-induced rise in cytosolic [Ca2+] involves Ca2+ ions from both extracellular and intracellular sources.A,Ca2+ signals were reduced, but not abolished, when Aβ42 oligomers (6 μg/ml) were applied to cells bathed in Ca2+-free medium. The trace shows the mean fluorescence (±1 S.E.) from 31 cells. B, depletion of endoplasmic reticulum Ca2+ stores does not abolish oligomer-induced Ca2+ signals in cells bathed in normal (1.8 mm Ca2+) medium. Following pretreatment with 5 μm thapsigargin, a specific blocker of sarcoplasmic/endoplasmic reticulum calcium ATPase Ca2+ pumps, applications of 6 μg/ml Aβ42 oligomers induced a large, rapid rise in fluorescence with an amplitude (ΔF/F = 0.7) similar to that obtained in the absence of thapsigargin (ΔF/F = 0.5). Trace shows mean ± 1 S.E. of results in 38 cells. C, Ca2+ signals evoked by Aβ42 oligomers are not reduced by cobalt, a nonspecific Ca2+ channel blocker. Cells were pretreated with 20 μm cobalt for 10 min. Subsequent application of 6 μg/ml of Aβ42 oligomers evoked a mean Ca2+-dependent fluorescence signal (black trace) of similar amplitude and kinetics to that in the absence of cobalt (gray trace). Data with cobalt were obtained from 27 cells. Control data (gray trace) are reproduced from Fig. 3A, trace c.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm the latter interpretation we then depleted the endoplasmic reticulum Ca2+ stores of cells bathed in normal [Ca2+] medium by applying thapsigargin, a specific sarcoplasmic/endoplasmic reticulum calcium ATPase pump inhibitor. Thapsigargin evoked a small rise in the Ca2+ signal, consistent with leakage of Ca2+ from intracellular stores into the cytosol, but subsequent application of Aβ42 oligomers produced a large, rapid increase, similar to that observed in control cells without thapsigargin treatment (Fig. 5B).We examined the possibility that amyloidogenic peptides may induce Ca2+ influx via endogenous Ca2+-permeable plasma membrane ion channels (30Guo Q. Furukawa K. Sopher B.L. Pham D.G. Xie J. Robinson N. Martin G.M. Mattson M.P. Neuroreport. 1996; 8: 379-38