From the
Departments of Cell & Molecular Biology and
Analytical Chemistry, Pharmacia Corporation, Kalamazoo, Michigan 49007
Received for publication, December 31, 2002 , and in revised form, April 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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A peptides are derived from consecutive processing of the amyloid precursor protein (APP) by two endopeptidases:
- and
-secretases. A membrane-bound aspartyl protease, named BACE1, was simultaneously identified as the
-secretase (1013). The molecular identity of
-secretase has not been fully revealed yet. Nevertheless, the transmembrane protein presenilin 1 seems indisputably required for the release of the amyloid peptide from its precursor (see reviews in Ref. 14). Pathogenetic studies have manifested that the majority of mutations in APP, presenilin 1, or presenilin 2, identified from the earlyonset familial AD patients, increase either total production of A
or the proportion of A
42 (reviewed in Ref. 15).
Monomeric A, when the critical concentration is reached, quickly folds into aggregated intermediate species such as oligomeric and protofibrillar forms, and finally, into insoluble fibrillar aggregates (16). Biophysical studies suggest that all these forms of A
are in equilibrium (1718). Increased production of A
peptides, particularly A
42, promotes amyloid fibrillogenesis and deposition in the limbic system (1920). In situ characterizations of brain tissues confirm the presence of aggregated A
fibrils (21) and SDS stable A
oligomers have been found in brains of Alzheimer patients (22). Earlier studies suggest that insoluble A
fibrils are toxic to neurons in vitro and are associated with neuronal damage in vivo (2326). Recently, studies using different approaches have demonstrated that both A
oligomers (2728) and protofibrils (2930) are neurotoxic as well.
During the optimization of substrates for measuring -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
-secretase site (31) and produced mutated (D1E/A2V) amyloid peptides that we named EV40 and EV42 to distinguish them from the wild-type A
40 and A
42. 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 A
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 A
40. We found that EV40 predominantly formed short, curvy, and sticky protofibrils that were similar to the intermediates of natural A
40 fibrils. Furthermore, this form of protofibrils was slightly more toxic to cultured SHEP cells and neurons than A
40. The work reported here has been the first to show that the N-terminal sequence of A
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 A
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MATERIALS AND METHODS |
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Turbidity Assays of Amyloid AggregationTurbidity assays were carried out as previously described (32). 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 MicroscopyPeptide 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 x2.5. Digital images were captured as TIFF files by a 1 megapixel Gatan Bioscan digital camera (model 792).
Circular Dichroism (CD) SpectroscopyPeptides 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 200260 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 (33), except -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 CultureHuman 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 NeuronsE18 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 x 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 AssaysCytotoxicity 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 x 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 ActivationFollowing 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 -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 x 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 x 104 cells, were electrophoresed on 412% 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).
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RESULTS |
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EV40 Predominantly Formed Protofibrils While A40 Assembled to Long FibrilsThe morphology of A
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 A
40. 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, A
40 formed long, rigid, and extended fibrils
510 nm in diameter that could be longer than 500 nm in length (Fig. 2A), consistent with the observations summarized previously (17). Under the same conditions, EV40 did not form long and rigid fibrils as seen in the A
40 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 A
protofibrils, identified as an intermediate that will progress to long and straight fibrils after longer incubation (17, 34). The EM morphology of EV40 also resembled the short protofibrils reported for a sample of A
40 in the presence of apomorphine (35). Thus, EV40 seemed to form an aggregated structure resembling the intermediate protofibrillar aggregates of natural A
40.
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We then conducted more careful studies focusing on the dynamic protofibrillar progression of EV40 and A40. Without incubation at 37 °C, no identifiable protofibrils or long fibrils were observed from freshly dissolved and negatively stained A
40 (Fig. 2C). Occasionally, we noticed a bead-like structure in freshly dissolved A
40 (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 A
. 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, A
40 proceeded to form short and irregular protofibrils (Fig. 2E), consistent with prior observations (17, 30, 34). 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 A
40 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. A40 always produced fibrillar aggregates no matter whether A
40 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 A40 by Circular Dichroism SpectroscopyTo determine the biophysical natures of A
40 and EV40 peptides, we employed circular dichroism to monitor the structural transition of these two peptides during aggregation. A solution of freshly prepared A
40-contained peptide in a mostly random coil and antiparallel
-sheet conformation as determined by principal component analysis of the CD spectrum, 47% random coil, 21%
-turns, 31% antiparallel
-sheet, <5% parallel
-sheet, and
-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 215230 nm as shown in Fig. 3A. Incubation for another 24 h produced additional spectral alterations consistent with increased antiparallel
-sheet.
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Unlike A40, 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 A
40 collected after 24 h at 37 °C. Similar conformation of EV40 with that of a protofibrillar aggregate of A
40 was consistent with the electron microscopy observations suggesting that EV40 formed an aggregated structure similar to the intermediate protofibrillar aggregates of A
40.
A comparison of CD spectra for A40 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
-sheet conformation was detected for A
40 upon incubation and although there was less random coil conformation in EV40 initially, there was less change in conformation after incubation. EV40 contained more
-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 A40Growth of A
40 fibrils can be affected by the presence of various reagents including short peptides (3536) and modification of residues within the A
region (37). To determine whether EV40 would interfere with the fibrillar assemblies of A
40, we mixed different ratios of A
40 to EV40 in the same tube and examined the morphological structures of the mixtures. When an equal amount of EV40 and A
40 powders were mixed in a tube and dissolved using the same procedure described for the above electron microscopy experiment, we found that the morphological structure of the mixture was more similar to EV40 preparations (Fig. 4A). The purities of each individual peptide and the mixed peptides used for the above EM experiments were verified by MALDI-TOF mass spectra (Fig. 4, BD). This suggested that EV40 might have suppressed fibrillation of A
40. However, this suppressive effect was not dominant since an increased proportion of A
40 (e.g. 80% of A
40) would increase the long fibrillar aggregates in the preparations (data not shown). Thus, EV40 could interfere with the assembly of A
40 into long fibrils.
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EV40 Is Slightly More Neurotoxic Than A40Previous experiments demonstrate that both A
protofibrils and fibrils induce acute electrophysiological changes and are toxic to cortical neurons (30). However, it was difficult to compare A
protofibrils and fibrils side by side due to limitations in the preparations of these materials. Here, we could compare the cellular toxicity of EV40 protofibrils with A
40 fibrils at the same concentrations. Prior to the treatment, we first incubated solutions of EV40 and A
40 at 37 °C for 48 h to allow aggregation, and then added the aggregated peptides to cultured human neuroblastoma SHEP cells. After cells were treated for 2 days, the cell viability was examined by assays using Sytox green nucleic acid fluorescent stains (3839). With the increase of either EV40 or A
40 concentrations, more cells showed toxicity (Fig. 5A, ** indicates p < 0.0001 and * indicates p < 0.005 relative to control). Cells treated with A
40 (50 µM) had
78% cell death, whereas the cells treated with the same concentration of EV40 showed
86% cell death (p = 0.2, A
40 versus EV40).
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We also compared cellular toxicity in SHEP cells treated with either freshly dissolved (without incubation as indicated with preaggregated) or aggregated (preincubation at 37 °C for 48 h) peptide solutions. Cells treated with freshly dissolved EV40 showed significantly higher cellular toxicity than A40 to SHEP cells (p < 0.0001, A
40 versus EV40), determined both by LDH release into the medium (Fig. 5B) as well as Sytox green fluorescence (Fig. 5C). This difference was clearly consistent with the EM experiments that the non-incubated A
40 presumably consists of both monomers and oligomers while EV40 contains mainly short protofibrils (see Fig. 2, C and D).
Addition of aggregated A40 into cultured SHEP cells showed a large increase in cellular toxicity compared with non-aggregated A
40, suggesting that SHEP cells were more vulnerable to aggregated A
40 than monomeric A
40. Further incubation of EV40 only slightly increased its cellular toxicity (Fig. 5, B and C), indicating that short protofibrils of EV40 were sufficient to kill SHEP cells. Altogether, these assays suggested that protofibrillar EV40 exhibited modest but consistently higher toxicity than fibrillar A
40 to SHEP cells in multiple independent experiments.
To confirm that EV40 is slightly more toxic to cells than A40, we repeated the study with cultured rat primary neurons prepared from the cortex or hippocampus. Similar to the observations in SHEP cells, we found that EV40 treatment caused an average of 85% death of rat primary neurons (p < 0.0001) while the same concentration of fibrillar A
40 resulted in 67% neuronal death on average (p < 0.001) (Fig. 5D). Hence, EV40 did appear to be more toxic, although not substantially, to both human neuronal cells and rat primary neurons when compared with the A
40 treatment.
Activation of Caspases by EV40 ProtofibrilsNeuronal toxicity induced by amyloid peptides has been partially attributed to programmed cell death (4041). Activation of caspase-2, -3, -6, -8, -9, and -12 in different neuronal cells by amyloid peptides have been demonstrated (4245). Although we observed cellular toxicity after SHEP cells were treated with either A40 or EV40, it was unclear whether this toxicity could be ascribed to apoptotic death. To explore this, we treated SHEP cells with either A
40 or EV40 in parallel and examined the activation of multiple caspases. Under our treatment conditions, we found that dramatic reductions of procaspase-9 with concomitant increases in the smaller active form were induced by either fibrillar A
40 or protofibrillar EV40 (Fig. 6), suggesting that both protofibrillar and fibrillar forms of amyloid peptides similarly activated caspases in the apoptotic pathway. We also observed similar activations of caspase-2, -3, and -8 by both A
40 and EV40 (data not shown). Likely, activation of caspase-9 occurred first and the activated caspase-9 in turn triggered sequential activation of downstream caspase-3, -2, and -8 (46). Apoptosis induced by protofibrillar amyloid species might implicate that AD neurons could be vulnerable to insult from intermediate amyloid aggregates, probably before the formation of amyloid plaques.
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DISCUSSION |
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Thioflavin T (ThT) fluorescence assay has also been used for determining the fibril formation. The fluorescent intensity of EV40 binding to ThT was smaller than that of A40 (data not shown), confirming that EV40 did not form long fibrils as A
40.
Studies of ex vivo amyloid fibrils from Alzheimer's brains provided clues to the structural organization of amyloid fibrils (48). However, these ex vivo studies provided only limited resolution of fibrillar structures. Synthetic amyloid peptides aggregated in vitro have been shown to mimic the fibrillar aggregates isolated from brain tissues. Furthermore, these studies also give rise to a better understanding of the dynamic assembly of amyloid fibrils. The assembly of amyloid fibrils has been shown to be a nucleation-dependent assembly process (4950). This nucleation requires non-covalent association of monomeric A, probably involving the conversion of
-helical or random coiled soluble A
structures to ordered
-structures (51). The arrangement of contiguous
-sheet A
peptides eventually produces short protofibrils and insoluble ribbon-like long fibrils. Preventing formation of stable
-pleated structure results in inhibiting aggregation of A
peptides (5152). Our CD spectra of EV40 indicated that EV40 could not lead to the transition from
-helical or random coiled structures to ordered
-structures during the incubation (Fig. 3), confirming that this transition is critical for the fibril formation.
Many factors including pH, temperature, salt, and changes of residues have been shown to affect the course of A fibrillogenesis (18, 53). Earlier studies focusing on the C terminus of A
show that residues 3442 are critical for the seeding and subsequent evolution of amyloid aggregates (19, 32, 5455). The central region of A
also plays a critical role in the progression to mature insoluble fibrils. Residues 1620 were identified to serve as a binding motif during polymerization (36). Studies of peptides containing Flemish (A21G), Arctic (E22G), and Dutch (E22Q) mutations suggest that these residues greatly affect the rate or the state of fibrillar aggregations (28, 34, 45, 5658). For example, Flemish A
40 (A21G) does not form protofibrils (34) while Arctic A
forms short and straight protofibrils (58). Previously, the N terminus of A
has been considered less crucial in affecting the course of fibrillogenesis. For example, N-terminally truncated A
(starting at Glu3, Phe4, Arg5, Gly8, Glu11, Val12, or Leu17) does not suppress, but instead enhances in some cases, aggregation in vitro (5960). In this study, we found that mutation of the first two amino acids of A
(D1E/A2V) had a profound effect on the course of amyloid fibrillogenesis as shown in Fig. 2 (A
40 versus EV40). Although peptides with the first few N-terminal residues deleted still form long amyloid fibrils as shown by Tekirian et al. (61), the presence of a bulky side chain at A
residue 2 could potentially alter the rate of protofibrillar extension as discussed in this study.
Nichols et al. (62) have suggested that growth of amyloid protofibrils can be achieved in two ways: elongation of protofibrils by monomer deposition and lateral association of protofibrils. An increase in NaCl concentration promotes protofibrillar extension via lateral association of protofibrils while addition of monomeric A favors elongation of protofibrils by monomer deposition. We found that EV40 forms smaller protofibrils almost immediately after the peptides were dissolved. It is likely that the concentration of monomeric EV40 became scarce shortly after the peptide was dissolved and this might suppress the elongation of EV40 protofibrils. Alternatively, the salt concentration was not optimal for promoting lateral association of EV40 protofibrils. Thus, more careful studies are necessary to gain insight into the mechanism of protofibrillar extension.
Although it still remains unclear how the bulkier Glu-Val side chains impact the structure of A in this region, the solubility or folding of this mutated peptide was at least altered. We found that both EV41 and EV42 could be synthesized by standard solid-phase methods, but could not be purified from the resin in two peptide synthesis facilities where A
41/A
42 are routinely synthesized and purified to homogeneity. Apparently, C-terminal extension of even one hydrophobic residue (Val) to this mutant peptide had a profound effect on the properties of EV40. It is likely that EV42 could be even more detrimental to cell growth considering the likelihood of its aggregation in the peptide synthesis resin. This could also explain our earlier observations that cells expressing high levels of APPISYEV, liberating EV40 and EV42, were unhealthy and prone to die when they reached moderately high confluency. Therefore, identification of EV40 may implicate that side chains of the N-terminal residues may affect assembly of fibrils. An antibody that recognizes an epitope within residues 36 of A
has already been shown to arrest the formation of A
fibrils and to dissolve already formed fibrils (63).
Moreover, the fibrillogenesis of EV40 apparently differs from Arctic A40. Arctic A
40 (E22G) was reported to increase the rate and quantity of protofibrillar species compared with wild-type A
40 (58). Nonetheless, Arctic A
40 continues to form long fibrillar species during longer incubation while EV40 did not form long and straight fibrils during extended incubation for up to 8 days.
One of the key issues in validating the "amyloid hypothesis" is whether A is detrimental to neurons. The proteinaceous components and compositions in the neuritic plaques are highly complicated. Technically, it is not possible yet to perform a neurotoxicity study by reconstituting in vivo situations using synthetic materials. Transgenic mice expressing only human APP variants fail to replicate AD pathological lesions (64). However, cellular toxicity studies using a single component (e.g. A
40 or A
42) have been extensively addressed to link A
fibrils to neuronal or synaptic loss seen in AD brains. A
in the range of 20100 µM has been shown to be toxic to neurons (2326), but no single mechanism can account for this induced neuronal death (66). In this study, we found that EV40 was moderately more toxic to both human neuroblastoma SHEP cells and rat primary neurons than wild type A
40. Hartley et al. (30) treated mixed brain cultures with protofibrillar A
40 that was prepared from size-exclusion chromatography and found that their protofibrillar A
40 was not as toxic as their long fibrillar A
40. This discrepancy could be related to the differences in initial doses of protofibrillar A
40 and fibrillar A
40 in their treatments. However, they demonstrate that protofibrillar A
40 causes increased frequency of action potentials and membrane depolarizations (30). Here we reported that multiple caspases in SHEP cells were activated by protofibrillar EV40. Since the protein levels of caspase-12 in SHEP cells are below our detection limit, we treated breast carcinoma MCF-7 cells with aggregated peptides and found that both protofibrillar EV40 and long fibrillar A
40 similarly activated caspase-12 (data not shown). Altogether, these results implicated that protofibrillar A
40 might play similar roles to fibrillar A
40 in affecting neuronal functions such as neurotransmitter release/uptake, long term potentiation, oxidative stress and neuronal survival. Consistent with this, A
oligomers have been shown to disrupt synaptic plasticity and cause neuronal toxicity (2728). Thus, blocking initial nucleation of A
, rather than dissolution of amyloid fibrils or inhibiting later stages of fibrillogenesis, may be a more relevant approach for AD treatment.
Thus far, clinical APP mutations at the N-terminal end of A region have not been identified yet. The data from this study suggested that a D1E/A2V mutation in the A
peptide could be severe. Mouse genetic studies with this mutation would provide valuable insight in this aspect as well.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Pharmacia Corporation, 301 Henrietta St., Kalamazoo, MI 49007. Tel.: 269-833-1450; Fax: 269-833-4255; E-mail: ryan{at}pharmacia.com.
1 The abbreviations used are: AD, Alzheimer's disease; APP, amyloid precursor protein; HPLC, high performance liquid chromatography; DMEM, Dulbecco's modified Eagle's medium.
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ACKNOWLEDGMENTS |
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REFERENCES |
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