Identification of a Mutant Amyloid Peptide That Predominantly Forms Neurotoxic Protofibrillar Aggregates
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m213298200
ISSN1083-351X
AutoresIsam Qahwash, Katherine L. Weiland, Yifeng Lu, Ronald W. Sarver, Rolf F. Kletzien, Riqiang Yan,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoThe amyloid peptide (Aβ), derived from the proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases, undergoes multistage assemblies to fibrillar depositions in the Alzheimer's brains. Aβ protofibrils were previously identified as an intermediate preceding insoluble fibrils. While characterizing a synthetic Aβ variant named EV40 that has mutations in the first two amino acids (D1E/A2V), we discerned unusual aggregation profiles of this variant. In comparison of the fibrillogenesis and cellular toxicity of EV40 to the wild-type Aβ peptide (Aβ40), we found that Aβ40 formed long fibrillar aggregates while EV40 formed only protofibrillar aggregates under the same in vitro incubation conditions. Cellular toxicity assays indicated that EV40 was slightly more toxic than Aβ40 to human neuroblastoma SHEP cells, rat primary cortical, and hippocampal neurons. Like Aβ40, the neurotoxicity of the protofibrillar EV40 could be partially attributed to apoptosis since multiple caspases such as caspase-9 were activated after SHEP cells were challenged with toxic concentrations of EV40. This suggested that apoptosis-induced neuronal loss might occur before extensive depositions of long amyloid fibrils in AD brains. This study has been the first to show that a mutated Aβ peptide formed only protofibrillar species and mutations of the amyloid peptide at the N-terminal side affect the dynamic amyloid fibrillogenesis. Thus, the identification of EV40 may lead to further understanding of the structural perturbation of Aβ to its fibrillation. The amyloid peptide (Aβ), derived from the proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases, undergoes multistage assemblies to fibrillar depositions in the Alzheimer's brains. Aβ protofibrils were previously identified as an intermediate preceding insoluble fibrils. While characterizing a synthetic Aβ variant named EV40 that has mutations in the first two amino acids (D1E/A2V), we discerned unusual aggregation profiles of this variant. In comparison of the fibrillogenesis and cellular toxicity of EV40 to the wild-type Aβ peptide (Aβ40), we found that Aβ40 formed long fibrillar aggregates while EV40 formed only protofibrillar aggregates under the same in vitro incubation conditions. Cellular toxicity assays indicated that EV40 was slightly more toxic than Aβ40 to human neuroblastoma SHEP cells, rat primary cortical, and hippocampal neurons. Like Aβ40, the neurotoxicity of the protofibrillar EV40 could be partially attributed to apoptosis since multiple caspases such as caspase-9 were activated after SHEP cells were challenged with toxic concentrations of EV40. This suggested that apoptosis-induced neuronal loss might occur before extensive depositions of long amyloid fibrils in AD brains. This study has been the first to show that a mutated Aβ peptide formed only protofibrillar species and mutations of the amyloid peptide at the N-terminal side affect the dynamic amyloid fibrillogenesis. Thus, the identification of EV40 may lead to further understanding of the structural perturbation of Aβ to its fibrillation. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; APP, amyloid precursor protein; HPLC, high performance liquid chromatography; DMEM, Dulbecco's modified Eagle's medium. 1The abbreviations used are: AD, Alzheimer's disease; APP, amyloid precursor protein; HPLC, high performance liquid chromatography; DMEM, Dulbecco's modified Eagle's medium. is the most common age-related neurodegenerative disorder. Extracellular amyloid plaques and intracellular neurofibrillary tangles are two typical pathological lesions of AD brains. Amyloid plaques, or neuritic plaques, which are more related to the AD pathogenesis, mainly consist of a cluster of heterogeneous amyloid peptides (Aβ) ranging from 39 to 43 amino acids (1Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 122: 1131-1135Crossref PubMed Scopus (1229) Google Scholar, 2Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3568) Google Scholar). Among these, the Aβ with 40 amino acids (Aβ40) accounts for about 90% while the less soluble C-terminally extended Aβ42 is close to 10%. The hyperphosphorylation of tau, a microtubule binding protein, leads to the formation of neurofibrillary tangles (3Grundke-Iqbal I. Iqbal K. Quinlan M. Tung Y.C. Zaidi M.S. Wisniewski H.M. J. Biol. Chem. 1986; 261: 6084-6089Abstract Full Text PDF PubMed Google Scholar, 4Ihara Y. Nukina N. Miura R. Ogawara M. J. Biochem. 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Nature. 2002; 416: 535-539Crossref PubMed Scopus (3630) Google Scholar) and protofibrils (29Walsh D.M. Hartley D.M. Kusumoto K. Fezoui Y. Lomakin A. Benedek G.B. Condron M.M. Selkoe D.J. Teplow D.B. J. Biol. Chem. 1999; 274: 25945-25952Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar, 30Hartley D.M. Walsh D.M. Ye C.P. Diehl T. Vasquez S. Vassilev P.M. Teplow D.B. Selkoe D.J. J. Neurosci. 1999; 19: 8876-8884Crossref PubMed Google Scholar) are neurotoxic as well. During the optimization of substrates for measuring β-secretase activity in cells, we generated several APP mutants including APPSYEV (K670S/M671Y/D672E/A673V) and APPisyev (V669I/K670S/M671Y/D672E/A673V). Expression of these two constructs in cells showed increased processing of mutant APP at the β-secretase site (31Tomasselli A.G. Qahwash I. Emmons T.L. Lu Y. Leone J.W. Lull J.M. Fok K.F. Bannow C.A. Smith C.W. Bienkowski M.J. Heinrikson R.L. Yan R. J. Neurochem. 2003; 84: 1006-1017Crossref PubMed Scopus (44) Google Scholar) and produced mutated (D1E/A2V) amyloid peptides that we named EV40 and EV42 to distinguish them from the wild-type Aβ40 and Aβ42. By examining the cell culture carefully, we found that stable cell lines expressing high levels of these two APP variants were less healthy than those expressing wild-type or Swedish APP. To understand whether the mutated residues in Aβ would change the properties of amyloid peptides and therefore cause the stress to the cell growth, we examined morphological structures and cellular toxicity of EV40 in comparison to Aβ40. We found that EV40 predominantly formed short, curvy, and sticky protofibrils that were similar to the intermediates of natural Aβ40 fibrils. Furthermore, this form of protofibrils was slightly more toxic to cultured SHEP cells and neurons than Aβ40. The work reported here has been the first to show that the N-terminal sequence of Aβ affected the rate and state of amyloid fibrillogenesis. This may lead to further exploration of the kinetics of amyloid aggregation affected by cellular factors that may potentially interact with the N-terminal end of Aβ. Peptide Synthesis—Amyloid β-(1–40) (Aβ40) was either purchased from Polypeptide Laboratories (Torrance, CA), or together with its mutant, EV40 (D1E/A2V), were synthesized by solid-phase methods employing an Applied Biosystems Model 433A Peptide Synthesizer. Crude peptide was dissolved in 0.05% trifluoroacetic acid, filtered and loaded onto a preparative reverse phase HPLC column (Vydac C-18, 22 × 250 mm, 10 micron) with a flow rate of 4 ml/min and equilibrated with solvent A (0.1% trifluoroacetic acid in water). The column was developed with a linear gradient employing solvents A and B (0.07% trifluoroacetic acid in acetonitrile): 0–10% B over 10 min and then 10–50% B over 100 or 200 min, depending on each individual peptide. The column eluent was monitored by absorbance at 220 and 280 nm. Fractions were monitored on an analytical reverse phase system (Vydac C18, 4.6 × 250 mm, 5 micron); solvents and conditions were as above. A linear gradient from 0–70% B over 20 min at 1.0 ml/min was employed for this purpose. The chemical authenticity of each peptide was established by mass spectrometry employing a Micromass Platform II mass spectrometer equipped with a Hewlett Packard Series 1050 HPLC system. The identity of the peptide was confirmed by injecting 5 μl of sample into the flow of 100 μl/min of 1:1 methanol/water. The mass spectrometer was operated in electrospray ionization mode with needle voltage 3 kV, temperature 120 °C, and cone voltage 30 V. The identity of the each peptide was confirmed by amino acid sequencing of the synthetic peptides. Turbidity Assays of Amyloid Aggregation—Turbidity assays were carried out as previously described (32Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1728) Google Scholar). Briefly,a1mm stock was made by dissolving the peptide in 0.22-μm filter-sterilized 0.1% acetic acid then diluted 1:20 in calcium and magnesium-free Dulbecco's phosphate buffered saline to a final concentration of 50 μm. Aliquots of the peptide solutions (250 μl) were transferred to wells of Corning 96-well tissue culture plates, and the wells were tightly sealed with an adhesive sealer. The plates were constantly shaken at 800 oscillations/min on a Titer Plate Shaker (Lab-Line Instruments, Inc., Melrose Park, IL) to induce aggregation. Monitoring the aggregation process was accomplished by measuring the optical density of each well at 405 nm using a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA). Electron Microscopy—Peptide solutions were prepared the same as for the turbidity assay to a final concentration of 50 μm. Peptide solutions were incubated at 37 °C for indicated times, and 5 μl of each sample was then applied onto formvar-coated copper grids (200 mesh) and negatively stained with 5 μl of 1% uranyl acetate for 1 min. Samples were viewed in a JEOL JEM-1230 transmission electron microscope at 80 kV accelerating voltage. Film images were captured on Kodak Electron Microscope film, and the negatives were developed and printed at an enlargement of ×2.5. Digital images were captured as TIFF files by a 1 megapixel Gatan Bioscan digital camera (model 792). Circular Dichroism (CD) Spectroscopy—Peptides were prepared by dissolving them in 0.22-μm filter-sterilized 0.1% acetic acid at 1 mm, the resulting solutions were then diluted 1:10 in 50 mm 0.2 μm filtered sodium phosphate buffer, pH 7.2 to a final concentration of 100 μm. Peptides were incubated at 37 °C for indicated times and aliquots of the peptide solutions (200 μl) were transferred to a quartz cell with a pathlength of 0.1 cm. Spectra were acquired using a Jasco (Easton, MD) J-715 spectrophotometer at 23 °C. The CD spectra were collected from 200–260 nm with a response of 0.25 s, scan speed of 100 nm/min, resolution of 1.0 nm and 32 cumulative scans. Secondary structures were estimated from the spectra using a principal component regression analysis method similar to one previously described (33Hennessey Jr., J.P. Johnson Jr., W.C. Biochemistry. 1981; 20: 1085-1094Crossref PubMed Scopus (573) Google Scholar), except β-turn structures were grouped together in the analysis and only 4 eigenvectors were used since the data did not extend to 178 nm. Error in the analysis of the basis spectra was within 5%. Stock solutions of both peptides were also prepared by dissolving the peptides directly in water at a concentration of 200 μm. These solutions were sampled immediately after preparation and after incubation at 37 °C for 72 h. Samples (15 μL) were transferred to a quartz cell with a pathlength of 0.01 cm, and CD spectra were acquired using the previous parameters except the data were collected from 184 to 260 nm and smoothed with a 13 point smoothing function. All CD spectra were converted to mean residue ellipticities for comparison. Cell Culture—Human SHEP cells are a cell line cloned from the neuroblastoma cell line SK-N-SH that was established in 1970 from a metastatic bone tumor. SHEP cells were maintained at 37 °C in a humidified, 5% CO2-controlled atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, and 2 mm glutamine. Preparation of Rat Primary Neurons—E18 pregnant rats were obtained from Charles River Laboratory and were anesthetized with halothane prior to cervical dislocation. Aseptically, embryos were removed and immersed in the dissociation media (Hibernate E supplemented with B27, 2 mm glutamine, and 50 units of penicillin/streptomycin). Embryo heads were cut away into a fresh 10-cm dish with dissociation medium. Overlying cartilage was dissected away, and the brains were moved to a fresh plate containing dissociation medium. Meninges and subcortical tissues were removed with fine forceps. Hippocampi and cortices were dissected and placed into 0.22 μm filter-sterilized 10 mg/ml papain/Hibernate solutions for 30 min at 35 °C with gentle agitation. Tissue was then washed twice with warm dissociation media and dispersed into a single cell suspension by gentle trituration through a fire-polished Pasteur pipette. Cells were counted on a Coulter Counter® ZM (Coulter Electronics, Luton Beds, England) and viability was determined by Trypan Blue exclusion. Cells were diluted in growth media (DMEM with 10% fetal bovine serum, 33 mm glucose, 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mm glutamine, B27, and 1 mm sodium pyruvate) and 1 × 105 cells/well were plated into poly-d-lysine coated 96-well tissue culture plates. On the fourth day of culture, half of the medium was replaced with growth media supplemented with aphidicolon to a final concentration of 3.3 μg/ml for 24 h after which the medium was replaced with growth media. Treatments were administered between days 12 and 14 in culture. Cellular Toxicity Assays—Cytotoxicity was quantified by assays based on penetration of Sytox Green Nucleic acid fluorescent stains into the damaged plasma membrane (Molecular Probes, Eugene, OR). Cell culture plates were centrifuged at 250 × g for 4 min. An aliquot of 50 μl of medium was removed from each well and replaced with 50 μl of a 2 μm Sytox Green solution, prepared by diluting the 5 mm stock in Me2SOwith phenol red-free Opti-MEM I or phenol red-free DMEM for SHEP cells or primary neurons, respectively. The plates were incubated for 30 min at 37 °C and read on a Tecan Spectrafluor Plus plate reader exciting at 485 nm and emitting at 535 nm. Alternatively, cellular toxicity was also determined by LDH assay (Promega, Madison, WI), measuring the release of the cytosolic lactate dehydrogenase at 492 nm. Western Blot Analysis of Caspase Activation—Following amyloid challenge, cells were harvested and lysed in MAPK lysis buffer on ice (20 mm HEPES (pH 7.3), 100 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm sodium orthovanadate, 0.1 mm molybdic acid, 10 mm MgCl2, 10 mm β-glycerophosphate, 5 mm p-nitrophenyl phosphate, 1 mm phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, 1 mm NaF, 5 μg/ml leupeptin, 5 μg/ml pepstatin, 0.05% 2-mercaptoethanol). The lysed cells were centrifuged at 10,000 × g for 10 min to remove cellular debris, and nuclei and cell lysates were then quantified based on protein concentrations. Equivalent protein samples, representing ∼7 × 104 cells, were electrophoresed on 4–12% Bis-Tris NuPAGE gel (Invitrogen, Carlsbad, CA). Following electrophoresis, proteins were transferred to an Immobilon-P membrane for Western analysis (Millipore, Bedford, MA). Processing/activation of caspases was evaluated by incubating with anti-actin antibody (0.5 μg/ml, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-caspase-9 (1:1000 dilution, PharMingen, San Diego, CA). Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), immunoreactivity was detected by chemiluminescence using SuperSignal West PICO reagent (Pierce). EV40 Displayed Different Aggregative Profiles from Aβ40 Based on Turbidimetric Assays—Aβ fibrillogenesis is a multistep process involving nucleation, elongation, and aggregation. The kinetics of Aβ fibrillogenesis has been previously monitored by various approaches including turbidimetry (32Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1728) Google Scholar). To gain a quick assessment of the EV40 aggregation, we compared kinetic aggregation profiles of EV40 to Aβ40 by turbidimetric assays. As shown in Fig. 1, after a short duration of lag phase (∼40 min) under constantly controlled shaking, Aβ40 solutions quickly became turbid. The peak of the turbidity, which reflects larger aggregation of the testing peptide, was reached at about 180-min postinitiation of shaking. Nevertheless, turbidity in the wells containing EV40 increased at a much slower rate (Fig. 1), suggesting that these two peptides aggregated with distinct kinetics of nucleation and oligomerization. At 360-min postinitiation of shaking, the maximum turbidity of the EV40-containing wells was significantly less than that of the Aβ40 containing wells. This observation implicated that both peptides had distinct aggregation rates, and, therefore, prompted us to examine the properties of EV40 more carefully. EV40 Predominantly Formed Protofibrils While Aβ40 Assembled to Long Fibrils—The morphology of Aβ fibrils has been well documented by the approaches of electron microscopy, atomic-force microscopy, etc. Since EV40 had a much slower aggregation rate based on the turbidimetric assays, we suspected that the biophysical parameters of EV40 might be different from Aβ40. To address this, we compared the morphological structures of these two peptides in parallel by electron microscopy. In our initial experiment, we allowed the peptides to aggregate at 37 °C for 48 h followed by examining the fibrillar formation. Under these conditions, Aβ40 formed long, rigid, and extended fibrils ∼5–10 nm in diameter that could be longer than 500 nm in length (Fig. 2A), consistent with the observations summarized previously (17Harper J.D. Wong S.S. Lieber C.M. Lansbury Jr., P.T. Biochemistry. 1997; 38: 8972-8980Crossref Scopus (454) Google Scholar). Under the same conditions, EV40 did not form long and rigid fibrils as seen in the Aβ40 preparations (comparing Fig. 2, A with B). Instead, the morphological structures found in the EV40 preparations were short and curvy with ∼5 nm in diameter (Fig. 2B), and ring-like structures were evident. Frequently, these short protofibrils tended to associate with each other, but did not assemble into long fibrils. In general, the morphological structure of EV40 was similar to that of the Aβ protofibrils, identified as an intermediate that will progress to long and straight fibrils after longer incubation (17Harper J.D. Wong S.S. Lieber C.M. Lansbury Jr., P.T. Biochemistry. 1997; 38: 8972-8980Crossref Scopus (454) Google Scholar, 34Walsh D.M. Lomakin A. Benedek G.B. Condron M.M. Selkoe D.J. Teplow D.B. J. Biol. Chem. 1997; 272: 22364-22372Abstract Full Text Full Text PDF PubMed Scopus (936) Google Scholar). The EM morphology of EV40 also resembled the short protofibrils reported for a sample of Aβ40 in the presence of apomorphine (35Lashuel 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 (142) Google Scholar). Thus, EV40 seemed to form an aggregated structure resembling the intermediate protofibrillar aggregates of natural Aβ40. We then conducted more careful studies focusing on the dynamic protofibrillar progression of EV40 and Aβ40. Without incubation at 37 °C, no identifiable protofibrils or long fibrils were observed from freshly dissolved and negatively stained Aβ40 (Fig. 2C). Occasionally, we noticed a bead-like structure in freshly dissolved Aβ40 (similar to that seen in the background of Fig. 2A). It is unclear whether it is related to the low molecular weight (LMW) oligomers of Aβ. On the contrary, EV40 formed smaller protofibrils (<100 nm in length) even without intentional incubation at 37 °C (Fig. 2D). If incubated for4hat 37 °C, Aβ40 proceeded to form short and irregular protofibrils (Fig. 2E), consistent with prior observations (17Harper J.D. Wong S.S. Lieber C.M. Lansbury Jr., P.T. Biochemistry. 1997; 38: 8972-8980Crossref Scopus (454) Google Scholar, 30Hartley D.M. Walsh D.M. Ye C.P. Diehl T. Vasquez S. Vassilev P.M. Teplow D.B. Selkoe D.J. J. Neurosci. 1999; 19: 8876-8884Crossref PubMed Google Scholar, 34Walsh D.M. Lomakin A. Benedek G.B. Condron M.M. Selkoe D.J. Teplow D.B. J. Biol. Chem. 1997; 272: 22364-22372Abstract Full Text Full Text PDF PubMed Scopus (936) Google Scholar). Incubation of the EV40 solution at 37 °C for 4 h would allow short protofibrillar EV40 to grow into longer protofibrils (Fig. 2F). Interestingly, even being incubated up to 8 days at 37 °C, EV40 still formed curvy protofibrillar aggregates and never formed long and straight fibrils (shown in Fig. 2G) while Aβ40 typically formed rigid fibrils after incubation for about 48 h (Fig. 2A). It appeared that EV40 had a very short duration of nucleation and oligomerization, but quickly locked into the protofibrillar state during prolonged incubation. Since synthetic peptides have been shown to have slight biophysical variations in batch-to-batch preparations, we repeated electron microscopy experiments using three different batches of EV40 to verify the above results. Again, we observed similar protofibrillar patterns using various batches of EV40, suggesting that the protofibrillar form of EV40 was not due to an unusual synthesis of this peptide. Aβ40 always produced fibrillar aggregates no matter whether Aβ40 peptide was purchased from a commercial source or synthesized in house employing the same preparative procedures for producing EV40. The Aggregative Progression Conformation of EV40 and Aβ40 by Circular Dichroism Spectroscopy—To determine the biophysical natures of Aβ40 and EV40 peptides, we employed circular dichroism to monitor the structural transition of these two peptides during aggregation. A solution of freshly prepared Aβ40-contained peptide in a mostly random coil and antiparallel β-sheet conformation as determined by principal component analysis of the CD spectrum, 47% random coil, 21% β-turns, 31% antiparallel β-sheet, <5% parallel β-sheet, and α-helical structure. Little change in the CD spectrum of the solution was detected for 2 h at room temperature but incubation at 37 °C for 24 h resulted in slightly greater negative ellipticity at 215–230 nm as shown in Fig. 3A. Incubation for another 24 h produced additional spectral alterations consistent with increased antiparallel β-sheet. Unlike Aβ40, a freshly prepared solution of EV40 showed little conformational change for 72 h at 37 °C (Fig. 3B). Interestingly, the CD spectra of EV40 were also very similar to the spectrum of Aβ40 collected after 24 h at 37 °C. Similar conformation of EV40 with that of a protofibrillar aggregate of Aβ40 was consistent with the electron microscopy observations suggesting that EV40 formed an aggregated structure similar to the intermediate protofibrillar aggregates of Aβ40. A comparison of CD spectra for Aβ40 and EV40 dissolved in water at 200 μm is shown in Fig. 3C. Initial spectra and spectra collected after 72 h incubation at 37 °C are shown. Using a shorter pathlength than used for the previous experiments, spectral data could be collected to lower wavelengths for these solutions that did not contain acetic acid or Me2SO that absorb at lower wavelengths. The same trend was evident as was detected at lower peptide concentrations. A large reduction in random coil with concomitant increase in β-sheet conformation was detected for Aβ40 upon incubation and although there was less random coil conformation in EV40 initially, there was less change in conformation after incubation. EV40 contained more β-sheet structure than AB40 initially as indicated by the ratio of ellipticities at 208 compared with 215 nm. Again, this is consistent with the other data that indicates EV40 forms protofibrils soon after dissolution but then undergoes a delay in elongated fibril formation. EV40 Suppresses Fibrillation of Aβ40—Growth of Aβ40 fibrils can be affected by the presence of various reagents including short peptides (35Lashuel H.A. Hartley D.M Balakhaneh D. Aggarwal A. Teichberg S. Callawa
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