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(The FASEB Journal. 2000;14:1233-1243.)
© 2000 FASEB

Fresh and globular amyloid ß protein (1–42) induces rapid cellular degeneration: evidence for AßP channel-mediated cellular toxicity

RAJINDER BHATIA12, HAI LIN2 and RATNESHWAR LAL

Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA

1Correspondence: Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA. E-mail: bhatia{at}lifesci.ucsb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Amyloid ß peptides (AßP) deposit as plaques in vascular and parenchymal areas of Alzheimer’s disease (AD) tissues and Down’s syndrome patients. Although neuronal toxicity is a feature of late stages of AD, vascular pathology appears to be a feature of all stages of AD. Globular and nonfibrillar AßPs are continuously released during normal cellular metabolism, form calcium-permeable channels, and alter cellular calcium level. We used atomic force microscopy, laser confocal microscopy, and calcium imaging to examine the real-time and acute effects of fresh and globular AßP1–42, AßP1–40, and AßP25–35 on cultured endothelial cells. AßPs induced morphological changes that were observed within minutes after AßP treatment and led to eventual cellular degeneration. Cellular morphological changes were most sensitive to AßP1–42. AßP1–42-induced morphological changes were observed at nanomolar concentrations and were accompanied by an elevated cellular calcium level. Morphological changes were prevented by anti-AßP antibody, AßP-channel antagonist zinc, and the removal of extracellular calcium, but not by tachykinin neuropeptide, voltage-sensitive calcium channel blocker cadmium, or antioxidants DTT and Trolox. Thus, nanomolar fresh and globular AßP1–42 induces rapid cellular degeneration by elevating intracellular calcium, most likely via calcium-permeable AßP channels and not by its interaction with membrane receptors or by activating oxidative pathways. Such rapid degeneration also suggests that the plaques, and especially fibrillar AßPs, may not have a direct causative role in AD pathogenic cascades.—Bhatia, R., Lin H., Lal, R. Fresh and globular amyloid ß protein (1–42) induces rapid cellular degeneration: evidence for AßP channel-mediated cellular toxicity


Key Words: AFM • scanning probe microscopy • real-time cellular imaging • endothelial cells • amyloid ß protein • neurotoxicity • Alzheimer’s disease • calcium imaging • cytoskeletal reorganization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
AMYLOID PLAQUES ARE present in the brain, cerebral blood vessels, and other aged tissues of patients with Alzheimer’s disease (AD) (1 , 2) . The plaques are made primarily of amyloid ß peptides, AßP1–42, and AßP1–40, products of proteolytic processing of a much larger amyloid precursor protein (APP), a ubiquitously expressed transmembrane glycoprotein. The level of these AßPs vary considerably among various forms of ADs; there is a differential accumulation of AßP1–40 and AßP1–42 in sporadic Alzheimer’s disease and nondemented brain samples (3) , and a mutation in presenilins is linked with an increased ratio of AßP1–42/AßP1–40 in familial Alzheimer’s disease (4 5 6 7) .

Macrovascular abnormalities often precede pathological features associated with AD (8 9 10) . AßP-induced inhibition of endothelial cell replication (11) and damage to both peripheral and cerebral vascular endothelium have been reported (12 , 13) . Consistent with cerebral vascular endothelial cell damage, a breach of the blood–brain barrier in AD is reported, which could allow entry of blood-borne AßP into the brain (14 15 16) .

Fibrillar plaques are found in AD tissues, however, their role in the etiology is uncertain. The in vitro degenerative effect of AßP has been examined, primarily after short-term treatment (3–4 h) with high concentrations (20–40 µM) of AßP or after long-term treatment (>24 h) with lower concentrations (~1 µM) of AßP (17 , 18) . The in vitro degenerative effect of AßP appears to correlate with its age and its fibrillar morphology (19) , though human AßP transgenic mice, which develop plaques, do not show comparable degeneration (20) . A recent study suggests that fibrillar forms of AßP may not be toxic and could even be cytoprotective (21) . Altered cellular properties and degeneration after a prolonged incubation with AßP may reflect a cascade of cellular responses, including altered gene expression and protein synthesis as well as the aging of the added AßPs.

Globular and nonfibrillar AßPs, which are continuously released during normal cellular metabolism, are also present in AD tissues. They form Ca2+-selective channels in reconstituted membrane, isolated plasma membrane, and in whole cell (22 23 24 25) and allow calcium uptake in reconstituted vesicles and can alter cellular calcium level (24 25 26 27) . Cellular responses mediated by these fresh globular proteins and AßP channels are yet to be determined. More specifically, AßP-induced short-term and localized microscopic changes in cytoskeletal organization are poorly understood. Such a lack of information is primarily because of the limited resolution of conventional light microscopy and the absence of a suitable method to examine local mechanical properties of living cells.

We have used an atomic force microscope (AFM) (for reviews, see ref 28 ) integrated with a fluorescence light microscope and calcium imaging with laser-scanning fluorescence microscopy to examine the real-time and acute effects of fresh and globular AßP1–42, AßP1–40, and AßP25–35 on cultured bovine aortic endothelial cells (BAEC). Endothelial cells were most sensitive to AßP1–42; the changes in cell morphology were observed at nanomolar concentrations of AßP1–42 and was accompanied by an increase in cellular Ca2+. AßP-induced cellular degeneration is also dependent on the presence of extracellular calcium. Moreover, the changes in cellular morphology after AßP treatment was blocked by zinc, previously shown to inhibit calcium uptake and conductance via membrane channels formed by AßP but not by voltage-sensitive calcium channel blocker cadmium. However, the AßP-induced changes in cellular morphology was not affected by Tachykinin neuropeptide or antioxidants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Cell culture
A BAEC line KOM-1 was obtained from Dr. Peter Davies at the University of Pennsylvania. Cells were cultured on sterile plastic petri dishes by a standard method in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose, 10 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated calf serum (Life Technologies, Rockville, Md.) (29) . Cells were maintained at 37°C and 5% CO2.

Chemicals and AßP treatment
ZnCl2, CdCl2, Tris, Trolox, DTT, and Physalaemin were purchased from Sigma-Aldrich (St. Louis, Mo.). AßP1–42, AßP1–40, and AßP25–35 peptides were obtained from Bachem (Torrance, Calif). AßP stock solutions were prepared by dissolving the peptides in deionized water. No DMSO or other solvents were used. The stock solutions were stored as aliquots at -20°C until used. AßPs were sonicated to disperse any fibrils before adding to the cells in culture or adsorbing on mica substrate for imaging single AßPs.

Immunofluorescence labeling
Mouse monoclonal anti-AßP antibody (3D6) against an epitope to the amino terminus (site 2–7) was a generous gift from Dr. Russel Rydel at Athena Neurosciences (South San Francisco, Calif.). Donkey anti-mouse-IgG conjugated with cy-3 was purchased from Chemicon (El Segundon, Calif.). Cells grown on glass coverslips were incubated with AßP (1 µM) for 30 min at 37°C, followed by a thorough wash with phosphate-buffered saline (PBS). Cells with and without AßP treatment were then fixed with 4% paraformaldehyde for 10 min and washed with PBS and PBS containing 3% bovine serum albumin and 1% donkey serum to minimize any nonspecific binding. Cells were then incubated with either 3D6 antibody against AßP (1 µg/ml) or normal mouse IgG (1 µg/ml) in PBS containing 3% BSA and 1% donkey serum for 1 h. After washing, the sample was incubated, for 1 h with a cy-3 conjugated donkey anti-mouse antibody (1:500 dilution) under the same conditions as for the primary antibody. Immunofluorescence images were captured with a Bio-Rad (Richmond, Calif.)MRC 1024 laser confocal microscope using a 60x Nikon PlanApo oil-immersion lens with 1.4 N.A.

Atomic force microscopy
AFM images were obtained as described (28 , 30) using a prototype of Bioscope AFM (Digital, Santa Barbara, Calif). Contact-mode AFM was used for most of the images. Oxide-sharpened silicon nitride tips, with a nominal spring constant of ~0.06 N/m (Digital), were used for most experiments. The imaging force was regularly monitored and kept to a minimum. The imaging force varied from a sub-nanonewton to tens of nanonewtons. All imaging was performed at room temperature (22–24°C).

Endothelial cells were imaged on days 2–3 after seeding. Cell monolayers cultured in plastic petri dishes were transferred into fresh DMEM (plus 20 mM HEPES) or HEPES-buffered OPTI-MEM-reduced serum medium (Life Technologies) so that the pH remained stable during AFM imaging. For the calcium-free condition, we used a specially ordered nominally calcium-free HEPES-buffered OPTI-MEM medium (Life Technologies), which contains no Ca2+ chelator, such as EGTA/EDTA. After optical alignment, AFM tip was lowered manually (under visual control with the integrated light microscope) onto an area with no cells. The imaging force was computed and minimized. Cells with well-defined morphology were then brought under the tip. With a scan size of ~30 x 30 nm, the force was again adjusted to a minimum. The scan size was gradually increased to the required size. The imaging force was regularly monitored and kept to a sub-nanonewton level so that no imaging-force-induced cellular deformations were observed (30) . The scan rate varied from 0.3 to 0.9 Hz (scan size 512x512 pixels). All imaging was performed at room temperature (22–24°C).

For control experiments (i.e., without AßP addition), cells were imaged often for >3 h and cells retained their viability and maintained stable morphology during that period. AFM images were continuously captured before cells were treated with any perturbation. A perturbation was then added online, and images were obtained continuously for another 2–3 h or until the cells lost viability. For each perturbation, the repeatability of the effect was imaged in at least 6–8 cell clusters in the same or different petri dish. The majority of AßP-induced cellular changes were observable within 35–45 min after the online addition of AßP.

Submicron-level structural changes are more easily observed at cellular edges and in the areas of cellular contacts (30) , and, given that the size of endothelial cells in our study was often larger than the AFM scan size limit, we primarily selected the cellular edges for presentation in this manuscript, although the structural changes were examined over the whole cell when possible.

Cell calcium imaging
Intracellular calcium level was imaged using a calcium-sensitive dye, Calcium Green-AM (Molecular Probes, Eugene, Oreg.), and a Bio-Rad MRC 1024 laser confocal microscope. Cells were cultured on glass coverslips (Fisher, Pittsburgh, Pa.) coated with collagen IV. To load the dye into the cells, cells were incubated with 5 µM Calcium Green-AM for 30–45 min at 37°C in PBS containing 1 mM Ca2+ and 1 mM Mg2+. The coverslip was then mounted into a chamber and placed on the stage of a Bio-Rad MRC-1024 laser confocal microscope. Intracellular calcium was imaged in cells incubated in HEPES-buffered OPTI-MEM-reduced serum medium (Life Technologies) at room temperature. For the calcium-free condition, we used a specially ordered nominally calcium-free HEPES-buffered OPTI-MEM medium (Life Technologies), which contains no Ca2+ chelator, such as EGTA/EDTA (which often leads to detachment of cells from coverslips). The excitation and emission wavelengths were selected at 488 and 515 nm, respectively. The objective used for the experiments was a 60x Nikon PlanApo oil-immersion lens with a numerical aperture of 1.4. The focal planes were set across the middle of cell bodies. Images were collected at 5 s intervals. The intracellular Ca2+ concentration was not calibrated for the present study.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Fresh AßPs are globular
Freshly prepared AßPs appear as discrete globular aggregates as imaged by AFM (Fig. 1 ). After storing at room temperature for 1 h, the time period during which AßPs induce irreversible cellular structural changes, < 1% of the AßPs formed short fibrillar aggregates and their size was small (<200 nm). However, after 24 h of incubation at room temperature, ~90% of the molecules formed large aggregates with a distinct fiber-like morphology (ref 25 and unpublished observation).



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  		Figure 1. AFM images of AßPs. Freshly prepared AßP1–42 appear as individual globular aggregates. A few large clumps are also visible that could be coagglomerates of globular particles. AßPs were thawed and adsorbed on freshly cleaved mica surface. After 10 min of adsorption, they were imaged under aqueous solution. In an identical set of experiments, after 1 h of incubation, <1% of the AßP1–42 forms larger fibrillar aggregates, but after 24 h of incubation, >90% assumes large fibrillar features (data not shown).

AßP1–42 induces cellular degeneration
We examined the short-term effects of AßP treatment on cell morphology and cell viability. Cellular morphological changes, including somal shrinkage, plasma membrane blebbing, and membrane rupture (as viewed in light microscopy and electron microscopy images), are commonly used as indicators for cellular degeneration (31 32 33) . An advantage of using AFM over other microscopic techniques for imaging living cells is that AFM allows imaging real-time cellular morphological changes on a submicron scale.

Cells not treated with AßP remained very stable and no significant cytoskeletal reorganization or morphological change was observed during 3 h or more of continuous imaging (Fig. 2a ,b ,c ,d ). Cells treated with 1 µM AßP1–42 showed changes in cellular morphology that began within 10–15 min of AßP treatment with a gradual but irreversible loss of cellular structure (Fig. 2e ,f ,g ,h ) and fragmentation of intracellular features, including plasma membranes organelles (Fig. 2f , star). The AßP-induced changes in cellular morphology were most dramatic at cellular peripheries, which occurs within 10 min of introduction of AßPs, in contrast to previous studies of AßPs toxicity after many hours of incubation. The short-term changes in central portions of cells were less pronounced compared with the peripheries. Some of the larger cellular retractions were even visible simultaneously under a light microscope and were consistent with previous observations in PC12 cells (33) . Such changes in cellular morphology were not imaging artifacts induced by the imaging force, because the imaging force can be extremely well controlled for nonperturbed imaging (30 ; for reviews see ref 28 ), and, in the present study, the morphological changes were apparent only in the AßP-treated and not in the untreated cells. Also, no difference in AßP effect was observed for cells incubated in either DMEM (plus 20 mM HEPES) or HEPES-buffered OPTI-MEM-reduced serum medium.



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  		Figure 2. Effect of AßP1–42 on morphological changes in endothelial cells. AFM image of the cells before treatment with AßPs is used as controls (time = 0). AßP1–42 was then added online and images captured continuously for another 2–3 h. Only the images taken at time 0 (control) and 30, 45, and 60 min are shown. a–d) Cells not treated with AßP did not show any appreciable morphological change (for example, see regions denoted by arrowheads) even after imaging for 3 h. e–h) Cells treated with 1 µM AßP1–42 show significant changes in cellular morphology compared with pre-AßP1–42 treatment (compare arrows in panels e–h). The changes in cellular morphology can be observed within 10–15 min after adding AßP1–42. The lengths of the arrows indicate the extent of retraction of cellular processes. In addition to a loss of peripheral structures, there is a loss of cytoskeletal structure as well (compare stars in panels f, e).

Such AßP-induced early cellular morphological changes, previously thought to occur only after many hours of AßP treatment, could signal the onset of AßP-induced cellular degeneration. In the present study, we did not directly examine AßP-induced cell death that could occur after prolonged incubation with AßPs. The most commonly used toxicity assay for detecting AßP-induced cell death, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, has been shown not to be a sufficient and specific measurement (34 , 35) . Furthermore, it is unclear if AßP-induced cell death is an apoptotic or necrotic process.

AßP1–42 is the most effective in inducing morphological changes
Cells treated with fresh AßP 1–42, AßP 1–40, or AßP25–35 exhibited significantly different levels of cellular degeneration (Fig. 3 ). Cells were most sensitive to AßP1–42. Incubation with nanomolar AßP1–42 induced rapid loss of cytoskeletal network, cell–cell connectivity, and sometimes complete detachment from the petri dish (Fig. 3g ,h ,i ). The changes in cellular morphology began within 10–15 min of incubation. At the same concentration, AßP25–35 and AßP1–40 did not induce any significant morphological changes even after 3 h of incubation (Fig. 3a ,b ,c ,d ,e ,f ). At higher concentrations, however, both AßP1–40 (10–20 µM) and AßP25–35 (40 µM) induced significant cellular degeneration (data not shown). AßP1–42 induced cellular degeneration in most cells (>90%). The repeatability of the effect was imaged in at least 6–8 cell clusters in the same or different petri dish. The majority of AßP-induced cellular changes were observable within 35–45 min after the online addition of AßP. In comparison with AßP1–42, AßP1–40 induced significantly greater cellular degeneration in cultured AD free aged human fibroblasts (36) . AßP1–40 is also reported to induce time- and concentration-dependent ultrastructural changes in PC12 cell membrane (33) .



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  		Figure 3. Effects of various AßPs on endothelial cell morphology. a–c) Cells treated with 1 µM AßP25–35; cells before (a) and 45 (b) and 60 (c) min after treatment with 1 µM AßP25–35. d–f) Cells before (d) and 45 (e) and 60 (f) min after treatment with 1 µM AßP1–40. No appreciable changes in cellular structure are observed for either AßP25–35 or AßP1–40 treatment (for example, see regions at periphery denoted by arrowheads). g–i) Cells treated with 50 nM AßP1–42; cells before (g) and 45 (h) and 60 (i) min after treatment with 50 nM AßP1–42; 50 nM AßP1–42 induced significant changes in cellular morphology (compare arrows in panels g–i). The lengths of the arrows indicate the extent of the retraction of the cellular processes. In addition to retractions of outer linings of cell membrane, there was also loss of cytoskeletal structures (compare stars in panels g–i). Only the AFM images taken before (time = 0, control) (left panels: a, d, g) and 45 and 60 min after treatment with different AßPs (middle and right panels: b, c, e, f, h, i) are shown.

We examined the effect of different concentrations of AßP1–42 (2 µM) (data not shown), 1 µM (Fig. 2e ,f ,g ,h ), 100 nM (Fig. 4a ,b ,c ), and 50 nM (Fig. 3g ,h ,i ) on endothelial cells. Based on our preliminary investigations, the rate of loss of cellular processes appears to be dependent on the concentration of AßP1–42, and the cellular degeneration is accelerated in the presence of higher levels of AßP1–42. The rate of cellular degeneration could also depend on other factors such as cell density, level of cellular communication, and temperature. A quantitative relationship between the extent of toxicity and the concentration of AßPs is currently under investigation.



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  		Figure 4. Specificity of AßP-induced changes in cell morphology. a–c) Effect of 100 nM AßP1–42. Images of cells before (a) and 45 (b) and 60 min (c) after treatment with 100 nM AßP1–42. A significant change in cellular morphology is visible (compare arrows in panel a with those in panels b, c). The lengths of the arrows indicate the extent of retraction of the cellular processes. d–f) Anti-AßP antibody (3D6) prevents cell degeneration. Images of cells before (d) and 45 (e) and 60 min (d) after treatment with 100 nM AßP1–42 in the presence of 20 µg/ml anti-AßP body (3D6). The overall cell morphology remained intact and no significant cytoskeletal reorganization occurred (compare arrowheads in panels d–f). Only the AFM images taken with and without anti-AßP antibody are shown.

Specificity of AßP effects
The specificity of AßP-induced cellular degeneration was examined using an anti-AßP antibody (3D6). When the anti-AßP antibody was added along with or immediately after the addition of AßP, the degenerative effect of AßP1–42 was nearly completely blocked; cell–cell connection was preserved and cells retained their normal morphology (Fig. 4d ,e ,f ). This provides strong evidence that the degeneration is specific to AßP treatment, and it also argues against any possible imaging artifacts, including tip- or imaging-force-induced cellular reorganizations.

Interaction of AßP1–42 with the cell membrane was also examined by immunofluorescence labeling using the 3D6 antibody. Cells preincubated with AßP1–42 exhibited strong immunofluorescence labeling on the plasma membrane surface (Fig. 5a ) but not in the interior of the cells. Cells not treated with AßP showed very little immunolabeling (Fig. 5c ). These results suggest that added AßP peptides were incorporated into the cell plasma membrane. Because the antibody used in our study is specific to the amino terminus portion of AßP, these results also suggest that the NH2 terminus of membrane-incorporated AßP1–42 is located outside the membrane. AFM images show that the plasma membrane of AßP-treated cells became rougher when incubated with anti-AßP-antibody; membrane ruffling was also visible after AßP treatment alone (data not shown) (36) . Such ruffling has been reported for AßP-transfected PC12 cells (37) .



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  		Figure 5. Laser confocal immunofluorescence imaging of cells treated with AßP1–42. The cells were incubated with 1 µM AßP1–42 for 30 min and immunolabeled with either a monoclonal anti-AßP antibody (3D6) (a) or normal mouse IgG (b) as a negative control, followed by cy-3 conjugated secondary antibody. c) Cells not treated with AßP1–42 exhibited little AßP immunofluorescence. All of these images are combined from two confocal planes (1 µM vertically apart) around the top cytoplasmic membrane.

In the present study, cells were fixed briefly with paraformaldehyde before immunolabeling. In a recent study, we have shown that in live fibroblast cells without fixation, AßP is also immunolocalized on the surface of AßP1–40-treated cells, suggesting that the antibody-binding epitope is located outside the cell membrane (36) . Anti-AßP immunolabeling has also been observed on cell membranes of paraformaldehyde-fixed AßP-treated neuronal cells (23) . In addition, we had reported AßP immunolocalization on the surface of nonpermeabilized lipid vesicle reconstituted with AßPs (24 , 25) .

Many cell types, including endothelial cells, are reported to express endogenous transmembrane APP and secrete soluble AßP (7) . A lack of strong immunolabeling in control cells not treated with AßP1–42 could be because of several reasons. Endothelial cells release very little, if any, soluble AßPs (7) . In the present study, cells were extensively washed before incubation with exogenous AßP1–42 and immunolabeling, which should remove endogenously released AßPs. Though these endothelial cells contain full-length transmembrane APPs, no previous immunolabeling with the 3D6 anti-AßP antibody has been reported, perhaps because the conformation of the endogenous APPs renders the antibody recognition site inaccessible.

AßP1–42 effect is mediated by calcium uptake via AßP pore
We examined several postulated mechanisms of AßP1–42-induced cytoskeletal reorganization which include 1) its interaction with the tachykinin receptors (38) , 2) AßP-induced enhanced responsiveness to oxidative stress (for review see ref 39 ), and 3) changing cellular ionic concentration (40 , 41) via opening and formation of ion channels (22 23 24 25 26 27) .

In the presence of physalaemin, a tachykinin, which should block the binding of AßP to potential tachykinin receptors (38) , AßP1–42 still induced significant changes in the cytoskeletal network and the loss of cell–cell contacts (Fig. 4a ,b ,c ), though prolonged incubation with physalaemin alone did not cause significant change in cell morphology. This suggests that AßP1–42-induced cytoskeletal reorganization is not mediated via its interaction with a previously proposed receptor pathway—the tachykinin neuropeptide pathway (38) . Physalaemin also does not prevent calcium-45 uptake in lipid vesicles reconstituted with AßP1–42 (24) . Physalaemin and other tachykinins are reported to modulate AßP-induced cytoskeletal reorganization in some neuronal cells (38) . Such difference in the action of tachykinins may reflect its effect to be cell-type dependent (neuronal vs. non-neuronal), though the reported effects of AßP are comparable and similar in both neuronal and non-neuronal cells.

It has also been proposed that AßPs induce cellular damage via the production of free radicals that presumably damages the cell plasma membrane. In the present study, antioxidants DTT and Trolox (a soluble analog of vitamin E) did not inhibit or retard the AßP1–42-induced cellular degeneration, which began within minutes after incubation with AßP1–42 (Figs. 6d ,e ,f ). This result suggests that the degeneration was a result of the oxidative stresses. All experiments in our study used freshly thawed and sonicated AßPs. These AßPs assume discrete globular structures as imaged by AFM (Fig. 1) . Recent studies suggest that AßPs do not spontaneously form peptide-derived free radicals (42) . In our study, morphological changes were rapid (within 10–15 min) and were observed with or without the presence of antioxidants. Thus, the AßP-induced cellular degeneration in these endothelial cells is unlikely to be mediated by the formation of free-radicals. Previous studies examining this mechanism have also produced conflicting results on cytoskeletal organization and cell lysis (19 , 32 , 43 44 45 46 47) .



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  		Figure 6. : AßP-induced changes in cellular morphology was blocked by Zn2+ and by the removal of extracellular calcium but not by tachykinin, antioxidants, and Cd2+. AFM images of endothelial cells taken before treatment with any perturbation (times = 0, controls) (left panels: a, d, g, j, m), 30 min (middle panels: b, e, h, k, n), and 45 min (right panels: c, f, i, l, o) after specific treatment are shown. a–c) Cells before (a) and 30 (b) and 45 min (c) after treatment with 1 µM AßP1–42 and 20 µM tachykinin (physalaemin). d–f) Cells before (d) and 30 (e) and 45 min (f) after treatment with 1 µM AßP1–42 in the presence of 500 µM Trolox. No protection from AßP-induced morphological changes was observed for the cells treated with tachykinin or Trolox (compare arrows in the control left panels with the middle and right panels). g–i) Cells before (g) and 30 (h) and 45 min (i) after treatment with 1 µM AßP1–42 in the presence of 100 µM CdCl2. CdCl2 did not protect cells from undergoing AßP-induced morphological changes (compare arrows in panels g–i). The lengths of the arrows indicate the extent of retraction of cellular processes. j–l) Cells before (j) and 30 (k) and 45 min (l) after treatment with 1 µM AßP1–42 in the presence of 50 µM ZnCl2. Zn2+ significantly protected cells from AßP-induced morphological changes (compare arrowheads in the panel j, control, with k, l). m–o) Cells before (m) and 30 (n) and 45 min (o) after treatment with 1 µM AßP1–42 in the absence of extracellular calcium. No appreciable changes in cell morphology are visible (compare arrowheads in the panels m–o).

An altered calcium homeostasis appears to be the common denominator underlying AßP-induced changes in cell morphology (41 , 48) . Soluble AßPs regulate cationic conductances and increase Ca2+ levels in AD and AD-free fibroblasts ( (40) Zhu et al., unpublished results), and neonatal hippocampal rat neurons (49) .

The AßP1–42-induced changes in cell morphology is also Ca2+-dependent. In a nominally Ca2+-free medium, AßP1–42 did not induce cellular degeneration (Fig. 6 , bottom panel). Moreover, zinc (50 µM) provided cells with very strong protection against AßP1–42-induced cellular degeneration (Fig. 6j ,k ,l ). Such protection from AßP-induced cellular degeneration was observed when zinc was added before, along with, or even 5 min after the addition of AßP1–42. Such a finding is consistent with earlier observations that AßPs form Ca2+-permeable pores in reconstituted vesicles and allow 45Ca2+ uptake that is blocked by Zn2+ and Tris (21 22 23 24 25 26) .

It is possible that the calcium uptake is via calcium-selective channels or large nonselective cationic pores present in the cell plasma membrane. In our study, Cd2+, which blocks voltage-sensitive Ca2+ channels, did not prevent AßP-induced morphological changes (Fig. 6g ,h ,i ), though a previous study has shown that AßP-induced Ca2+ currents in tetracarcinoma cells are inhibited by CdCl2. (50) . Previous studies from other laboratories have also shown that several calcium channel blockers and antagonists of calcium mobilization, such as {omega}-conotoxin, nifedipine, verapamil, APV, MK-801, cAMP, 8-bromo cAMP, and cGMP, did not inhibit neurotoxicity induced by AßP25–35 and AßP1–40 (51) . Thus, although it is difficult to exclude all possibilities of calcium uptake through a modulation of existing channels, the inhibition of cellular degeneration by presently available specific blockers of AßP channel activity, zinc and Tris, but not by calcium channel blocker cadmium, strongly suggest that AßP toxicity is mediated by calcium uptake via AßP channels. Recent studies have demonstrated the formation of relatively nonselective ion channels in planar phospholipid bilayer by amylin, PrP 106–126, and AßP (22 23 24 25 26 , 52 53 54 55) . The mechanism of cell and tissue destruction or dysfunction in amyloid diseases has been postulated to be mediated via these channels.

We examined changes in the intracellular Ca2+ level using a Ca2+-sensitive dye Calcium Green and a Bio-Rad MRC 1024 laser confocal microscope. Application of 0.22 µM AßP1–42-induced transient (10–20 s in duration) and often repetitive increases of Ca2+ (Ca2+ waves) with no apparent synchronization in 43% of the cells (Fig. 7a , I–V). At 0.44 µM, AßP1–42 induced Ca2+ waves in 73% of cells (Fig. 7a , I–VIII) and at higher frequency. When AßP1–42 concentration was raised to 2.2 µM, Ca2+ level increased simultaneously in nearly all cells (Figs. 7a , I–X; Fig. 8j ), even in cells that did not respond to 0.22 or 0.44 µM AßP1–42 (Fig. 7a , IX and X). In 23% of the cells, addition of 2.2 µM AßP1–42 induced a sustained increase in calcium, followed by a slow decrease in calcium (Fig. 7a , VIII and X); whereas in 77% of the cells, after the transient increase in the Ca2+ fluorescence, the fluorescence levels rapidly dropped to near zero ({Delta}F/F0 = 1) within 30 s, indicating the dye had leaked out of the cells. These results indicate that high levels of AßP1–42 cause rapid cell degeneration and damages in the cell plasma membranes.



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  		Figure 7. : Calcium levels in representative BAECs in response to different concentrations of AßP1–42 in the presence (a) or absence (b) of extracellular calcium. The data in each plot in this figure represent the relative change of the average calcium green fluorescence intensity of a single cell, {Delta}F/F0 = (F-F0)/F0, where F is the average fluorescence intensity of a specific cell and F0 is the same average intensity before AßP1–42 treatment. {Delta}F/F0 = 1 indicates no change in fluorescence level and {Delta}F/F0 = -1 means F = 0. Arrows above the plots indicate when AßP1–42 (0.22, 0.44, and 2.2 µM) was added. In the presence of extracellular calcium (a), 0.22 and 0.44 µM AßP1–42 induced Ca2+ waves in 43 and 73%, respectively; 2.2 µM AßP1–42 caused a sharp drop fluorescence intensity in 77% of the cells, after a transient increase, to near zero ({Delta}F/F0 {approx} -1 or F {approx} 0), indicating the fluorescent dye had leaked out of the cells. In the nominally Ca2+-free medium, only 1 cell out of 38 cells examined exhibited any oscillation of Ca2+ (data not shown) in response to 0.22, 0.44, or 2.2 µM of AßP1–42; 2.2 µM of AßP1–42 also did not cause any leakage of dye from the cells.



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  		Figure 8. Modified representative confocal calcium green fluorescence images of endothelium cells before and after AßP1–42 treatments as plotted in Fig. 7 . The color-coded average pixel calcium green fluorescence pixel intensities, {Delta}F/F0=(F-F0)/F0, were superimposed on low-intensity background images of the cells, where F is the current pixel fluorescence intensity and F0 is the pixel intensity immediately before Ca2+ increase occurred. a) Before AßP1–42 was added. b–e) Representative snapshots of Ca2+ waves in some cells after 0.22 µM AßP1–42. f–i) Representative snapshots of Ca2+ waves in some cells after 0.44 µM AßP1–42. j) Immediately after the addition of 2.2 µM AßP1–42, Ca2+ level increased sharply in all cells. k) 6 min after addition of 2.2 µM AßP1–42, most cells became completely dark due to loss of dye.

AßP1–42-induced Ca2+ increase/Ca2+ wave is dependent on the presence of extracellular Ca2+ (Fig. 7b ). When cells were incubated in a nominally Ca2+-free medium, only 1 out of 38 cells examined showed any Ca2+ oscillation in response to AßP1–42 (data not shown). Also, in the nominally Ca2+-free medium, 2.2 µM AßP1–42 did not cause any leakage of fluorescent dye, indicating that AßP1–42-induced damage to cytoplasmic membrane was dependent on the presence of extracellular Ca2+, consistent with the AFM images of morphological changes.

Agonist-induced elevation of intracellular Ca2+ could be initiated by and composed of coordinated elementary events of Ca2+ signals, such as Ca2+ ‘sparks’ and Ca2+ ‘puffs’ (56) . These elementary events could represent Ca2+ released from intracellular stores by the activation of inositol 1,4,5-tris-phosphate receptors (IP3Rs) or ryanodine receptors (RYRs). Opening of plasma membrane Ca2+ channels could also produce Ca2+ ‘puffs’, which can create regenerative Ca2+ waves or a sustained elevated Ca2+ level. Because AßP1–42-induced dose-dependent Ca2+ waves require extracellular Ca2+, the main source of elevated Ca2+ is the influx from the extracellular medium and not the release from internal Ca2+ stores (which is often activated via a receptor mediated pathway).

The Ca2+-imaging data are consistent with the hypothesis that AßP1–42 forms Ca2+-permeable channels in endothelial cell plasma membrane, in a dose-dependent manner, and Ca2+ influx via single AßP channels produce elementary Ca2+ signals, which leads to Ca2+ waves at lower concentrations and sustained Ca2+ increase at higher concentrations (Figs. 7 , 8) . The Ca2+ imaging experiments also show that AßP1–42 (at 2.2 µM) causes rapid cellular damage, particularly to the cytoplasmic membrane, which is unlikely to be caused by an oxidative mechanism.

Our results thus show that the AßP-induced cellular degeneration is initiated by calcium uptake via AßP. A localized calcium change could alter local micromechanical properties (13) and induce cytoskeletal reorganization. Indeed, disruption of the cytoskeleton is one of the earliest detectable changes that correlates with neurodegenerative disorders such as AD (1) . The dose-dependent relationship between AßP and internal calcium level suggests that a smaller increase in the internal calcium, which could result from the presence of a relatively small number of AßP channels formed by nominally released soluble AßPs, could be compensated for by the calcium-buffering mechanisms of cells. However, enhanced production and/or decreased removal of soluble AßPs could result in a considerably larger number of AßP channels. These channels, in turn, would allow increased levels of calcium uptake, possibly beyond the buffering capacity of cells, which could lead to a cascade of cellular pathological events. Designing new blockers/inhibitors and/or screening potential blockers/inhibitors of AßP channels thus could provide new effective therapeutic avenues to prevent cellular damage caused by AßP.


   ACKNOWLEDGMENTS
 
We thank Drs. Seung Rhee, Arjan Quist, and Nils Almqvist for insightful advice and suggestions, and Maura Jess for help in the preparation of figures. We thank Dr. Ashok Parbhu for providing preliminary data on AßP fibrillogenesis. We thank Dr. Russell Rydel from Athena Neurosciences for kindly providing us with the anti-AßP antibodies. The work was supported by the Alzheimer’s Disease Program, Department of Health, California, and NIH (GM-NIA). We are grateful to the anonymous reviewers for their useful suggestions to improve the quality and relevance of our results. Portions of this work have been presented at the American Society for Cell Biologists Annual Meeting, San Francisco, December 1998.


   FOOTNOTES
 
2 These two authors contributed equally.

Received for publication July 16, 1999. Revised for publication October 7, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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