bathia 2000 toxicity 1.42

8/3/2019 Bathia 2000 Toxicity 1.42

1/11

Fresh and globular amyloid protein (142) inducesrapid cellular degeneration: evidence for APchannel-mediated cellular toxicity

RAJINDER BHATIA,1,2

HAI LIN,2

RATNESHWAR LALNeuroscience Research Institute, University of California, Santa Barbara, California 93106, USA

ABSTRACT Amyloid peptides (AP) deposit asplaques in vascular and parenchymal areas of Alzhei-mers disease (AD) tissues and Downs syndrome

patients. Although neuronal toxicity is a feature oflate stages of AD, vascular pathology appears to be afeature of all stages of AD. Globular and nonfibrillar

APs are continuously released during normal cellu-lar metabolism, form calcium-permeable channels,and alter cellular calcium level. We used atomicforce microscopy, laser confocal microscopy, andcalcium imaging to examine the real-time and acuteeffects of fresh and globular AP142, AP140, and

AP2535 on cultured endothelial cells. APs in-duced morphological changes that were observed

within minutes after AP treatment and led to even-tual cellular degeneration. Cellular morphologicalchanges were most sensitive to AP142. AP142-induced morphological changes were observed atnanomolar concentrations and were accompanied byan elevated cellular calcium level. Morphologicalchanges were prevented by anti-AP antibody, AP-

channel antagonist zinc, and the removal of extracel-lular calcium, but not by tachykinin neuropeptide,

voltage-sensitive calcium channel blocker cadmium,or antioxidants DTT and Trolox. Thus, nanomolarfresh and globular AP142 induces rapid cellulardegeneration by elevating intracellular calcium, mostlikely via calcium-permeable AP channels and notby its interaction with membrane receptors or byactivating oxidative pathways. Such rapid degenera-tion also suggests that the plaques, and especiallyfibrillar APs, may not have a direct causative role in

AD pathogenic cascades.Bhatia, R., Lin H., Lal, R.

Fresh and globular amyloidprotein (142) inducesrapid cellular degeneration: evidence for AP chan-nel-mediated cellular toxicityFASEB J. 14, 12331243(2000)

Key Words: AFM scanning probe microscopy real-time cel-lular imaging endothelial cells amyloid protein neuro-toxicity Alzheimers disease calcium imaging cytoskeletalreorganization

Amyloid plaques are present in the brain, cerebralblood vessels, and other aged tissues of patients with

Alzheimers disease (AD) (1, 2). The plaques aremade primarily of amyloid peptides, AP142, andAP140, products of proteolytic processing of amuch larger amyloid precursor protein (APP), aubiquitously expressed transmembrane glycopro-tein. The level of these APs vary considerablyamong various forms of ADs; there is a differentialaccumulation of AP140 and AP142 in sporadic

Alzheimers disease and nondemented brain sam-ples (3), and a mutation in presenilins is linked withan increased ratio of AP142/AP140 in familialAlzheimers disease (4 7).

Macrovascular abnormalities often precede patho-logical features associated with AD (810). AP-induced inhibition of endothelial cell replication(11) and damage to both peripheral and cerebralvascular endothelium have been reported (12, 13).Consistent with cerebral vascular endothelial celldamage, a breach of the bloodbrain barrier in ADis reported, which could allow entry of blood-borne

AP into the brain (1416).Fibrillar plaques are found in AD tissues, however,their role in the etiology is uncertain. The in vitrodegenerative effect of AP has been examined,primarily after short-term treatment (34 h) withhigh concentrations (2040 M) of AP or afterlong-term treatment (24 h) with lower concentra-tions (1 M) of AP (17, 18). The in vitro degen-erative effect of AP appears to correlate with its ageand its fibrillar morphology (19), though humanAP transgenic mice, which develop plaques, do notshow comparable degeneration (20). A recent study

suggests that fibrillar forms of A

P may not be toxicand could even be cytoprotective (21). Altered cel-lular properties and degeneration after a prolongedincubation with AP may reflect a cascade of cellularresponses, including altered gene expression andprotein synthesis as well as the aging of the addedAPs.

1 Correspondence: Neuroscience Research Institute, Uni-versity of California, Santa Barbara, CA 93106, USA. E-mail:[email protected]

2 These two authors contributed equally.

12330892-6638/00/0014-1233/$02.25 FASEB


2/11

Globular and nonfibrillar APs, which are contin-uously released during normal cellular metabolism,are also present in AD tissues. They form Ca2-selective channels in reconstituted membrane, iso-lated plasma membrane, and in whole cell (2225)and allow calcium uptake in reconstituted vesiclesand can alter cellular calcium level (2427). Cellularresponses mediated by these fresh globular proteinsand AP channels are yet to be determined. More

specifically, AP-induced short-term and localizedmicroscopic changes in cytoskeletal organization arepoorly understood. Such a lack of information isprimarily because of the limited resolution of con- ventional light microscopy and the absence of asuitable method to examine local mechanical prop-erties of living cells.

We have used an atomic force microscope (AFM)(for reviews, see ref 28) integrated with a fluores-cence light microscope and calcium imaging withlaser-scanning fluorescence microscopy to examinethe real-time and acute effects of fresh and globular

A

P142, A

P140, and A

P2535 on cultured bovineaortic endothelial cells (BAEC). Endothelial cellswere most sensitive to AP142; the changes in cellmorphology were observed at nanomolar concentra-tions of AP142 and was accompanied by an in-crease in cellular Ca2. AP-induced cellular degen-eration is also dependent on the presence ofextracellular calcium. Moreover, the changes in cel-lular morphology after AP treatment was blockedby zinc, previously shown to inhibit calcium uptakeand conductance via membrane channels formed byAP but not by voltage-sensitive calcium channelblocker cadmium. However, the AP-induced

changes in cellular morphology was not affected byTachykinin neuropeptide or antioxidants.

MATERIALS AND METHODS

Cell culture

A BAEC line KOM-1 was obtained from Dr. Peter Davies at theUniversity of Pennsylvania. Cells were cultured on sterileplastic petri dishes by a standard method in Dulbeccosmodified Eagles medium (DMEM) containing glucose, 10mM 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 main-tained at 37C and 5% CO2.

Chemicals and AP treatment

ZnCl2, CdCl2, Tris, Trolox, DTT, and Physalaemin werepurchased from Sigma-Aldrich (St. Louis, Mo.). AP142,

AP140, and AP2535 peptides were obtained from Bachem(Torrance, Calif). AP stock solutions were prepared bydissolving the peptides in deionized water. No DMSO orother solvents were used. The stock solutions were stored asaliquots at20C until used. APs were sonicated to disperse

any fibrils before adding to the cells in culture or adsorbingon mica substrate for imaging single APs.

Immunofluorescence labeling

Mouse monoclonal anti-AP antibody (3D6) against anepitope to the amino terminus (site 27) was a generous giftfrom Dr. Russel Rydel at Athena Neurosciences (South SanFrancisco, Calif.). Donkey anti-mouse-IgG conjugated withcy-3 was purchased from Chemicon (El Segundon, Calif.).

Cells grown on glass coverslips were incubated with AP (1M) for 30 min at 37C, followed by a thorough wash withphosphate-buffered saline (PBS). Cells with and without APtreatment were then fixed with 4% paraformaldehyde for 10min and washed with PBS and PBS containing 3% bovineserum albumin and 1% donkey serum to minimize anynonspecific binding. Cells were then incubated with either3D6 antibody against AP (1 g/ml) or normal mouse IgG (1g/ml) in PBS containing 3% BSA and 1% donkey serum for1 h. After washing, the sample was incubated, for 1 h with acy-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 us-ing a 60 Nikon PlanApo oil-immersion lens with 1.4 N.A.

Atomic force microscopy

AFM images were obtained as described (28, 30) using aprototype 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 constantof0.06 N/m (Digital), were used for most experiments. Theimaging force was regularly monitored and kept to a mini-mum. The imaging force varied from a sub-nanonewton totens of nanonewtons. All imaging was performed at roomtemperature (2224C).

Endothelial cells were imaged on days 23 after seeding.Cell monolayers cultured in plastic petri dishes were trans-

ferred into fresh DMEM (plus 20 mM HEPES) or HEPES-buffered OPTI-MEM-reduced serum medium (Life Technol-ogies) so that the pH remained stable during AFM imaging.For the calcium-free condition, we used a specially orderednominally calcium-free HEPES-buffered OPTI-MEM medium(Life Technologies), which contains no Ca2 chelator, suchas EGTA/EDTA. After optical alignment, AFM tip was low-ered manually (under visual control with the integrated lightmicroscope) onto an area with no cells. The imaging force

was computed and minimized. Cells with well-defined mor-phology were then brought under the tip. With a scan size of30 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 asub-nanonewton level so that no imaging-force-induced cel-

lular deformations were observed (30). The scan rate variedfrom 0.3 to 0.9 Hz (scan size 512512 pixels). All imaging wasperformed at room temperature (2224C).

For control experiments (i.e., without AP addition), cellswere imaged often for 3 h and cells retained their viabilityand maintained stable morphology during that period. AFMimages were continuously captured before cells were treated

with any perturbation. A perturbation was then added online,and images were obtained continuously for another 23 h oruntil the cells lost viability. For each perturbation, the repeat-ability of the effect was imaged in at least 68 cell clusters inthe same or different petri dish. The majority of AP-inducedcellular changes were observable within 3545 min after theonline addition of AP.

1234 Vol. 14 June 2000 BHATIA ET AL.The FASEB Journal


3/11

Submicron-level structural changes are more easily ob-served 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 primarilyselected the cellular edges for presentation in this manu-script, although the structural changes were examined overthe whole cell when possible.

Cell calcium imaging

Intracellular calcium level was imaged using a calcium-sensi-tive 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 M Calcium Green-AM for 3045min at 37C in PBS containing 1 mM Ca2 and 1 mM Mg2.The coverslip was then mounted into a chamber and placedon the stage of a Bio-Rad MRC-1024 laser confocal micro-scope. Intracellular calcium was imaged in cells incubated inHEPES-buffered OPTI-MEM-reduced serum medium (LifeTechnologies) at room temperature. For the calcium-freecondition, we used a specially ordered nominally calcium-freeHEPES-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 488and 515 nm, respectively. The objective used for the experi-ments was a 60 Nikon PlanApo oil-immersion lens with anumerical aperture of 1.4. The focal planes were set acrossthe middle of cell bodies. Images were collected at 5 sintervals. The intracellular Ca2 concentration was not cali-brated for the present study.

RESULTS AND DISCUSSION

Fresh APs are globular

Freshly prepared APs appear as discrete globularaggregates as imaged by AFM (Fig. 1). After storingat room temperature for 1 h, the time period during which APs induce irreversible cellular structuralchanges, 1% of the APs formed short fibrillaraggregates and their size was small (200 nm).However, after 24 h of incubation at room tempera-ture, 90% of the molecules formed large aggre-gates with a distinct fiber-like morphology (ref 25and unpublished observation).

AP142 induces cellular degeneration

We examined the short-term effects of AP treat-ment on cell morphology and cell viability. Cellularmorphological changes, including somal shrinkage,plasma membrane blebbing, and membrane rupture(as viewed in light microscopy and electron micros-copy images), are commonly used as indicators forcellular degeneration (3133). An advantage of us-ing AFM over other microscopic techniques forimaging living cells is that AFM allows imagingreal-time cellular morphological changes on a sub-micron scale.

Cells not treated with AP remained very stableand no significant cytoskeletal reorganization ormorphological change was observed during 3 h ormore of continuous imaging (Fig. 2a d). Cells

treated with 1 M AP142 showed changes in cellu-lar morphology that began within 1015 min of APtreatment with a gradual but irreversible loss ofcellular structure (Fig. 2e h) and fragmentation ofintracellular features, including plasma membranesorganelles (Fig. 2f, star). The AP-induced changesin cellular morphology were most dramatic at cellu-lar peripheries, which occurs within 10 min of intro-duction of APs, in contrast to previous studies ofAPs toxicity after many hours of incubation. Theshort-term changes in central portions of cells wereless pronounced compared with the peripheries.

Some of the larger cellular retractions were evenvisible simultaneously under a light microscope andwere consistent with previous observations in PC12cells (33). Such changes in cellular morphology werenot imaging artifacts induced by the imaging force,because the imaging force can be extremely wellcontrolled for nonperturbed imaging (30; for reviews see ref 28), and, in the present study, themorphological changes were apparent only in theAP-treated and not in the untreated cells. Also, nodifference in AP effect was observed for cells incu-bated in either DMEM (plus 20 mM HEPES) or

Figure 1. AFM images of APs. Freshly prepared AP142appear as individual globular aggregates. A few large clumpsare also visible that could be coagglomerates of globularparticles. APs were thawed and adsorbed on freshly cleavedmica surface. After 10 min of adsorption, they were imagedunder aqueous solution. In an identical set of experiments,after 1 h of incubation, 1% of the AP142 forms largerfibrillar aggregates, but after 24 h of incubation, 90%assumes large fibrillar features (data not shown).

1235AP142

INDUCES CA2-DEPENDENT RAPID CELLULAR DEGENERATION


4/11

HEPES-buffered OPTI-MEM-reduced serum me-dium.

Such AP-induced early cellular morphologicalchanges, previously thought to occur only after many

hours of AP treatment, could signal the onset ofAP-induced cellular degeneration. In the presentstudy, we did not directly examine AP-induced celldeath that could occur after prolonged incubation withAPs. The most commonly used toxicity assay fordetecting AP-induced cell death, the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay, has been shown not to be a sufficient and specificmeasurement (34, 35). Furthermore, it is unclear ifAP-induced cell death is an apoptotic or necroticprocess.

AP142 is the most effective in inducingmorphological changes

Cells treated with fresh AP 142, AP 140, or AP2535 exhibited significantly different levels of cellu-lar degeneration (Fig. 3). Cells were most sensitive toAP142. Incubation with nanomolar AP142 in-duced rapid loss of cytoskeletal network, cellcellconnectivity, and sometimes complete detachmentfrom the petri dish (Fig. 3gi). The changes incellular morphology began within 1015 min ofincubation. At the same concentration, AP2535 and

AP140 did not induce any significant morphologi-cal changes even after 3 h of incubation (Fig. 3af). At higher concentrations, however, both AP140(1020 M) and AP2535 (40 M) induced signifi-

cant cellular degeneration (data not shown).AP142 induced cellular degeneration in most cells(90%). The repeatability of the effect was imagedin at least 68 cell clusters in the same or differentpetri dish. The majority of AP-induced cellularchanges were observable within 3545 min after theonline addition of AP. In comparison with AP142,AP140 induced significantly greater cellular degen-eration in cultured AD free aged human fibroblasts(36). AP140 is also reported to induce time- andconcentration-dependent ultrastructural changes inPC12 cell membrane (33).

We examined the effect of different concentra-tions of AP142 (2 M) (data not shown), 1 M(Fig. 2e h), 100 nM (Fig. 4a c), and 50 nM (Fig.3gi) on endothelial cells. Based on our preliminaryinvestigations, the rate of loss of cellular processesappears to be dependent on the concentration ofAP142, and the cellular degeneration is acceler-ated in the presence of higher levels of AP142. Therate of cellular degeneration could also depend onother factors such as cell density, level of cellularcommunication, and temperature. A quantitativerelationship between the extent of toxicity and the

Figure 2. Effect of AP142 on morphological changes in endothelial cells. AFM image of the cells before treatment with APsis used as controls (time 0). AP142 was then added online and images captured continuously for another 23 h. Only theimages taken at time 0 (control) and 30, 45, and 60 min are shown. ad) Cells not treated with AP did not show any appreciablemorphological change (for example, see regions denoted by arrowheads) even after imaging for 3 h. eh)Cells treated with 1M AP142 show significant changes in cellular morphology compared with pre-AP142 treatment (compare arrows in panelse h). The changes in cellular morphology can be observed within 1015 min after adding AP142. The lengths of the arrowsindicate the extent of retraction of cellular processes. In addition to a loss of peripheral structures, there is a loss of cytoskeletalstructure as well (compare stars in panels f, e).



5/11

concentration of APs is currently under investiga-tion.

Specificity of AP effects

The specificity of AP-induced cellular degeneration was examined using an anti-AP antibody (3D6).When the anti-AP antibody was added along with or

immediately after the addition of AP, the degener-ative effect of AP142 was nearly completelyblocked; cellcell connection was preserved andcells retained their normal morphology (Fig. 4df).This provides strong evidence that the degenerationis specific to AP treatment, and it also arguesagainst any possible imaging artifacts, including tip-or imaging-force-induced cellular reorganizations.

Interaction of AP142 with the cell membrane wasalso examined by immunofluorescence labeling us-ing the 3D6 antibody. Cells preincubated withAP142 exhibited strong immunofluorescence la-

beling on the plasma membrane surface (Fig. 5a)but not in the interior of the cells. Cells not treated with AP showed very little immunolabeling (Fig.5c). These results suggest that added AP peptideswere incorporated into the cell plasma membrane.Because the antibody used in our study is specific tothe amino terminus portion of AP, these resultsalso suggest that the NH2 terminus of membrane-incorporated AP142 is located outside the mem-

brane. AFM images show that the plasma membraneof AP-treated cells became rougher when incu-bated with anti-AP-antibody; membrane rufflingwas also visible after AP treatment alone (data notshown) (36). Such ruffling has been reported forAP-transfected PC12 cells (37).

In the present study, cells were fixed briefly withparaformaldehyde before immunolabeling. In a re-cent study, we have shown that in live fibroblast cellswithout fixation, AP is also immunolocalized on thesurface of AP140-treated cells, suggesting that theantibody-binding epitope is located outside the cell

membrane (36). Anti-A

P immunolabeling has alsobeen observed on cell membranes of paraformalde-hyde-fixed AP-treated neuronal cells (23). In addi-tion, we had reported AP immunolocalization onthe surface of nonpermeabilized lipid vesicle recon-stituted with APs (24, 25).

Many cell types, including endothelial cells, arereported to express endogenous transmembrane APP and secrete soluble AP (7). A lack of strongimmunolabeling in control cells not treated withAP142 could be because of several reasons. Endo-thelial cells release very little, if any, soluble APs(7). In the present study, cells were extensively

washed before incubation with exogenous AP142and immunolabeling, which should remove endog-enously released APs. Though these endothelialcells contain full-length transmembrane APPs, noprevious immunolabeling with the 3D6 anti-APantibody has been reported, perhaps because theconformation of the endogenous APPs renders theantibody recognition site inaccessible.

AP142 effect is mediated by calcium uptake viaAP pore

We examined several postulated mechanisms ofAP142-induced cytoskeletal reorganization whichinclude 1) its interaction with the tachykinin recep-tors (38), 2) AP-induced enhanced responsivenessto oxidative stress (for review see ref 39), and 3)changing cellular ionic concentration (40, 41) viaopening and formation of ion channels (2227).

In the presence of physalaemin, a tachykinin,which should block the binding of AP to potentialtachykinin receptors (38), AP142 still induced sig-nificant changes in the cytoskeletal network and the

Figure 3. Effects of various APs on endothelial cell morphol-ogy. a c) Cells treated with 1 M AP2535; cells before (a)and 45 (b) a n d 6 0 (c) min after treatment with 1 M

AP2535. df) Cells before (d) and 45 (e) and 60 (f) min aftertreatment with 1 M AP140. No appreciable changes incellular structure are observed for either AP2535 or AP140treatment (for example, see regions at periphery denoted byarrowheads). gi) Cells treated with 50 nM AP142; cellsbefore (g) and 45 (h) and 60 (i) min after treatment with 50nM AP142; 50 nM AP142 induced significant changes incellular morphology (compare arrows in panels gi). Thelengths of the arrows indicate the extent of the retraction ofthe cellular processes. In addition to retractions of outerlinings of cell membrane, there was also loss of cytoskeletalstructures (compare stars in panels gi). Only the AFMimages taken before (time 0, control) (left panels: a, d, g)and 45 and 60 min after treatment with different APs(middle and right panels: b, c, e, f, h, i) are shown.

1237AP142



6/11

loss of cellcell contacts (Fig. 4a c), though pro-longed incubation with physalaemin alone did notcause significant change in cell morphology. This

suggests that AP142-induced cytoskeletal reorgani-zation is not mediated via its interaction with apreviously proposed receptor pathwaythe tachyki-

Figure 4. Specificity of AP-induced changes in cell morphology. ac) Effect of 100 nM AP142. Images of cells before (a) and45 (b) and 60 min (c) after treatment with 100 nM AP142. A significant change in cellular morphology is visible (comparearrows in panel awith those in panels b, c). The lengths of the arrows indicate the extent of retraction of the cellular processes.df) Anti-AP antibody (3D6) prevents cell degeneration. Images of cells before (d) and 45 (e) and 60 min (d) after treatment

with 100 nM AP142 in the presence of 20 g/ml anti-AP body (3D6). The overall cell morphology remained intact and nosignificant cytoskeletal reorganization occurred (compare arrowheads in panels df). Only the AFM images taken with and

without anti-AP antibody are shown.

Figure 5. Laser confocal immunofluorescence imaging of cells treated with AP142. The cells were incubated with 1 MAP142 for 30 min and immunolabeled with either a monoclonal anti-AP antibody (3D6) (a) or normal mouse IgG (b) as anegative control, followed by cy-3 conjugated secondary antibody. c) Cells not treated with AP142 exhibited little APimmunofluorescence. All of these images are combined from two confocal planes (1 M vertically apart) around the topcytoplasmic membrane.



7/11

nin neuropeptide pathway (38). Physalaemin alsodoes not prevent calcium-45 uptake in lipid vesiclesreconstituted with AP142 (24). Physalaemin andother tachykinins are reported to modulate AP-induced cytoskeletal reorganization in some neuro-nal cells (38). Such difference in the action oftachykinins may reflect its effect to be cell-typedependent (neuronal vs. non-neuronal), though thereported effects of AP are comparable and similar

in both neuronal and non-neuronal cells.It has also been proposed that APs induce cellu-

lar damage via the production of free radicals thatpresumably damages the cell plasma membrane. Inthe present study, antioxidants DTT and Trolox (asoluble analog of vitamin E) did not inhibit or retardthe AP142-induced cellular degeneration, whichbegan within minutes after incubation with AP142(Figs. 6df). This result suggests that the degenera-tion was a result of the oxidative stresses. All exper-iments in our study used freshly thawed and soni-cated APs. These APs assume discrete globular

structures as imaged by AFM (Fig. 1). Recent studiessuggest that APs do not spontaneously form pep-tide-derived free radicals (42). In our study, morpho-logical changes were rapid (within 1015 min) andwere observed with or without the presence of anti-oxidants. Thus, the AP-induced cellular degenera-tion in these endothelial cells is unlikely to bemediated by the formation of free-radicals. Previousstudies examining this mechanism have also pro-duced conflicting results on cytoskeletal organiza-tion and cell lysis (19, 32, 4347).

An altered calcium homeostasis appears to be the

common denominator underlying AP-inducedchanges in cell morphology (41, 48). Soluble APsregulate cationic conductances and increase Ca2

levels in AD and AD-free fibroblasts ( (40) Zhu et al.,unpublished results), and neonatal hippocampal ratneurons (49).

The AP142-induced changes in cell morphologyis also Ca2-dependent. In a nominally Ca2-freemedium, AP142 did not induce cellular degenera-tion (Fig. 6, bottom panel). Moreover, zinc (50 M)provided cells with very strong protection againstAP142-induced cellular degeneration (Fig. 6jl).

Such protection from AP-induced cellular degen-eration was observed when zinc was added before,along with, or even 5 min after the addition ofAP142. Such a finding is consistent with earlierobservations that APs form Ca2-permeable poresin reconstituted vesicles and allow 45Ca2 uptakethat is blocked by Zn2 and Tris (2126).

It is possible that the calcium uptake is via calcium-selective channels or large nonselective cationicpores present in the cell plasma membrane. In ourstudy, Cd2, which blocks voltage-sensitive Ca2

channels, did not prevent AP-induced morpholog-

Figure 6. : AP-induced changes in cellular morphology wasblocked by Zn2 and by the removal of extracellularcalcium but not by tachykinin, antioxidants, and Cd2.

AFM images of endothelial cells taken before treatmentwith 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) a n d 3 0 (b) and 45 min (c) aftertreatment with 1 M AP142 and 20 M tachykinin(physalaemin). df) Cells before (d) and 30 (e) and 45 min(f) after treatment with 1 M AP142 in the presence of500 M Trolox. No protection from AP-induced morpho-logical changes was observed for the cells treated withtachykinin or Trolox (compare arrows in the control left

panels with the middle and right panels). gi) Cells before(g) and 30 (h) and 45 min (i) after treatment with 1 MAP142 in the presence of 100 M CdCl2. CdCl2 did notprotect cells from undergoing AP-induced morphologicalchanges (compare arrows in panels gi). The lengths of thearrows indicate the extent of retraction of cellular processes. jl)Cells before (j) and 30 (k) and 45 min (l) after treatment with 1M AP142 in the presence of 50 M ZnCl2. Zn

2 significantlyprotected cells from AP-induced morphological changes (com-pare arrowheads in the panel j, control, with k, l). mo) Cellsbefore (m) and 30 (n) and 45 min (o) after treatment with 1 M

AP142 in the absence of extracellular calcium. No appreciablechanges in cell morphology are visible (compare arrowheads inthe panels mo).

1239AP142



8/11

ical changes (Fig. 6gi), though a previous study hasshown that AP-induced Ca2 currents in tetracarci-

noma cells are inhibited by CdCl2. (50). Previousstudies from other laboratories have also shown thatseveral calcium channel blockers and antagonists ofcalcium mobilization, such as -conotoxin, nifedi-pine, verapamil, APV, MK-801, cAMP, 8-bromocAMP, and cGMP, did not inhibit neurotoxicityinduced by AP2535 and AP140 (51). Thus, al-though it is difficult to exclude all possibilities ofcalcium uptake through a modulation of existingchannels, the inhibition of cellular degeneration bypresently available specific blockers of AP channelactivity, zinc and Tris, but not by calcium channel

blocker cadmium, strongly suggest that AP toxicityis mediated by calcium uptake via AP channels.Recent studies have demonstrated the formation ofrelatively nonselective ion channels in planar phos-pholipid bilayer by amylin, PrP 106126, and AP(2226, 5255). The mechanism of cell and tissuedestruction or dysfunction in amyloid diseases hasbeen postulated to be mediated via these channels.

We examined changes in the intracellular Ca2

level using a Ca2-sensitive dye Calcium Green and aBio-Rad MRC 1024 laser confocal microscope. Appli-cation of 0.22 M AP142-induced transient (1020

s in duration) and often repetitive increases of Ca2

(Ca2 waves) with no apparent synchronization in

43% of the cells (Fig. 7a, IV). At 0.44 M, AP142induced Ca2 waves in 73% of cells (Fig. 7a, IVIII)and at higher frequency. When AP142 concentra-tion was raised to 2.2 M, Ca2 level increasedsimultaneously in nearly all cells (Figs. 7a, IX; Fig.8j), even in cells that did not respond to 0.22 or 0.44M AP142 (Fig. 7a, IX and X). In 23% of the cells,addition of 2.2 M AP142 induced a sustainedincrease in calcium, followed by a slow decrease incalcium (Fig. 7a, VIII and X); whereas in 77% of thecells, after the transient increase in the Ca2 fluores-cence, the fluorescence levels rapidly dropped to

near zero (F/F0 1) within 30 s, indicating the dyehad leaked out of the cells. These results indicatethat high levels of AP142 cause rapid cell degener-ation and damages in the cell plasma membranes.

AP142-induced Ca2 increase/Ca2 wave is de-

pendent 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 showedany Ca2 oscillation in response to AP142 (data notshown). Also, in the nominally Ca2-free medium,2.2 M AP142 did not cause any leakage of fluo-rescent dye, indicating that AP142-induced dam-

Figure 7. : Calcium levels in representative BAECs inresponse to different concentrations of AP142 in thepresence (a) or absence (b) of extracellular calcium. Thedata in each plot in this figure represent the relativechange of the average calcium green fluorescence intensityof a single cell, F/F0 (F-F0)/F0, where F is the averagefluorescence intensity of a specific cell and F

0is the same

average intensity before AP142 treatment. F/F0 1indicates no change in fluorescence level and F/F01means F 0. Arrows above the plots indicate when

AP142 (0.22, 0.44, and 2.2 M) was added. In thepresence of extracellular calcium (a), 0.22 and 0.44 MAP142 induced Ca

2 waves in 43 and 73%, respectively;2.2M AP142 caused a sharp drop fluorescence intensityin 77% of the cells, after a transient increase, to near zero(F/F01 or F 0), indicating the fluorescent dye hadleaked out of the cells. In the nominally Ca2-free medium,only 1 cell out of 38 cells examined exhibited any oscilla-tion of Ca2 (data not shown) in response to 0.22, 0.44, or2.2 M of AP142; 2.2 M of AP142 also did not causeany leakage of dye from the cells.



9/11

age to cytoplasmic membrane was dependent on thepresence of extracellular Ca2, consistent with the

AFM images of morphological changes. Agonist-induced elevation of intracellular Ca2

could be initiated by and composed of coordinatedelementary events of Ca2 signals, such as Ca2

sparks and Ca2 puffs (56). These elementaryevents could represent Ca2 released from intracel-lular stores by the activation of inositol 1,4,5-tris-phosphate receptors (IP3Rs) or ryanodine receptors(RYRs). Opening of plasma membrane Ca2 chan-nels could also produce Ca2 puffs, which cancreate regenerative Ca2 waves or a sustained elevated Ca2 level. Because AP142-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 frominternal Ca2 stores (which is often activated via areceptor mediated pathway).

The Ca2-imaging data are consistent with thehypothesis that AP142 forms Ca

2-permeablechannels in endothelial cell plasma membrane, in adose-dependent manner, and Ca2 influx via singleAP channels produce elementary Ca2 signals, which leads to Ca2 waves at lower concentrationsand sustained Ca2 increase at higher concentra-tions (Figs. 7, 8). The Ca2 imaging experimentsalso show that AP142 (at 2.2 M) causes rapid

Figure 8. Modified representative confocal calcium green fluores-cence images of endothelium cells before and after AP142

treatments as plotted in Fig. 7. The color-coded average pixelcalcium green fluorescence pixel intensities, F/F0(F-F0)/F0,were superimposed on low-intensity background images of thecells, whereFis the currentpixel fluorescence intensity andF

0is the

pixel intensity immediately before Ca2 increase occurred. a)Before AP142 was added. be) Representative snapshots of Ca

2

waves in some cells after 0.22 M AP142. fi) Representativesnapshots of Ca2 waves in some cells after 0.44 M AP142. j)Immediately after the addition of 2.2 M AP142, Ca

2 levelincreased sharply in all cells. k) 6 min after addition of 2.2 M

AP142, most cells became completely dark due to loss of dye.

1241AP142



10/11

cellular damage, particularly to the cytoplasmicmembrane, which is unlikely to be caused by anoxidative mechanism.

Our results thus show that the AP-inducedcellular degeneration is initiated by calcium up-take via AP. A localized calcium change couldalter local micromechanical properties (13) andinduce cytoskeletal reorganization. Indeed, dis-ruption of the cytoskeleton is one of the earliest

detectable changes that correlates with neuro-degenerative disorders such as AD (1). The dose-dependent relationship between AP and internalcalcium level suggests that a smaller increase inthe internal calcium, which could result from thepresence of a relatively small number of APchannels formed by nominally released solubleAPs, could be compensated for by the calcium-buffering mechanisms of cells. However, en-hanced production and/or decreased removal ofsoluble APs could result in a considerably largernumber of AP 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 patholog-ical events. Designing new blockers/inhibitorsand/or screening potential blockers/inhibitors ofAP channels thus could provide new effectivetherapeutic avenues to prevent cellular damagecaused by AP.

We thank Drs. Seung Rhee, Arjan Quist, and Nils Almqvistfor insightful advice and suggestions, and Maura Jess for helpin the preparation of figures. We thank Dr. Ashok Parbhu forproviding preliminary data on AP fibrillogenesis. We thankDr. Russell Rydel from Athena Neurosciences for kindlyproviding us with the anti-AP antibodies. The work wassupported by the Alzheimers Disease Program, Departmentof Health, California, and NIH (GM-NIA). We are grateful tothe anonymous reviewers for their useful suggestions toimprove the quality and relevance of our results. Portions ofthis work have been presented at the American Society forCell Biologists Annual Meeting, San Francisco, December1998.

REFERENCES

1. Mattson, M. P. (1997) Cellular actions of beta-amyloid precursorprotein and its soluble and fibrillogenic derivatives. Physiol. Rev.77, 10811132

2. Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C.,Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H.,Schenk, D., Teplow, D. B., and Selkoe, D. J. (1992) Amyloid-peptide is produced by cultured cells during normal metabo-lism. Nature (London) 359, 322325

3. Nashlund, J., Thyberg, J., Tjernberg, L. O., Wernstedt, C.,Karlstrom, A. R., Bogdanovic, N., Gandy, S. E., Lannfelt, L.,Terenius, L., and Nordstedt, C. (1995) Characterization ofstable complexes involving apolipoprotein E and the amyloidbeta peptide in Alzheimers disease brain. Neuron 15, 219222

4. Antonsson, B., Conti, F., Ciavatta, A., Montessuit, S., Lewis, S.,Martinou, I., Bernasconi, L., Benard, A., Mermod, J.-J., Mazzei,G., Maundrell, K., Gambale, F., Sadoul, R., and Martinou. J.-C.

(1997) Inhibition of Bax channel-forming activity by Bcl-2.Science 277, 370372

5. Duff, K., Eckman, C., Zher, C., Yu, X., Prada, C. M., Perez-Tur,J., Hutton, M., Buee, L., Harigaya., Y., Yager, D., Morgan, D.,Gordon, M. N., Holcomb, L., Refolo, L., Zenk, B., Hardy, J., andYounkin, S. (1996) Increased amyloid 42(43) in brains of miceexpressing mutant presenilin 1. Nature (London) 383, 710713

6. Schendel, A. L., Xie, Z., Montal, M. O., Matsuyama, S., Montal,M., and Reed, J. C. (1997) Channel formation by antiapoptoticprotein Bcl-2. Proc. Natl. Acad. Sci. USA 94, 51135118

7. Scheuner, D., Eckman, C., Jenson, M., Song, X., Citron, M.,Suzuki, N., Bird, T. D., Hardy, J., Hutton, M., Kukull, W.,

Larson, E., Levy-Lahard, E., Vitanen, M., Peskind, E., Poorkar,P., Schellenberg, G., Tanzi, R., Wasco, W., Lanfelt, L., Selkoe,D., and Younkin. S. (1996) Secreted amyloid--protein similar tothat in the senile plaques of Alzheimers disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked tofamilial Alzheimers disease. Nat. Med. 2, 864870

8. Buee, L., Hof, P. R., Bouras, C., Delacourte, A., Perl, D. P.,Morrison, J. H., and Fillit, H. M. (1994) Pathological alterationsof the cerebral microvasculature in Alzheimers disease andrelated dementing disorders. Acta Neuropath. 87, 469480

9. Busciglio, J., Gabuzda, D. H., Matsu, P., and Yankner, B. A.(1993) Generation of beta-amyloid in the secretory pathway inneuronal and non-neuronal cells. Proc. Natl. Acad. Sci. USA 90,20922096

10. Perry, G., Smith, M. A., McCann, C. E., Siedlak, S. L., Jones, P. K.,and Friedland, R. P. (1998) Cerebrovascular muscle atrophy is afeature of Alzheimers Disease. Brain Res. 791, 6366

11. Grammas, P., Botchlet, T., Fugate, R., Ball, M. J., and Roher, A. E. (1995) Alzheimer disease amyloid proteins inhibit brainendothelial cell proliferation in vitro. Dementia 6, 126130

12. Crawford, F., Suo, Z., Fang, C., and Mullan, M. (1998) Charac-teristics of the in vitro vasoactivity of -amyloid peptides. Exp.Neurol. 150, 159168

13. Thomas, T., Thomas, G., McLendon, C., Sutton, T., and Mullan,M. (1996) -Amyloid-mediated vasoactivity and vascular endo-thelial damage. Nature (London) 380, 168171

14. Mattila, K. M., Pirttila, T., Blennow, K., Wallin, A., Viitanen, M.,and Frey, H. (1994) Altered blood-brain-barrier function inAlzheimers disease? Acta Neurol. Scand. 89, 192198

15. Mecocci, P., Parnetti, L., Romano, G., Scarelli, A., Chionne, F.,Cecchetti, R., Polidori, M. C., Palumbo, B., Cherubini, A., andSenin, U. (1995) Serum anti-GFAP and anti-S100 autoantibod-ies in brain aging. Alzheimers disease and vascular dementia.

J. Neuroimmunol. 57, 16517016. Perlmutter, L. S. (1994) Microvascular pathology and vascularbasement membrane components in Alzheimers disease. Mol.Neurobiol. 9, 3340

17. Blanc, E. M., Toborek, M., Mark, R. J., Henning, B., and Mattson,M. P. (1997) Amyloid -peptide induces cell monolayer albuminpermeability, impairs glucose transport, and induces apoptosis invascular endothelial cells. J. Neurochem. 68, 18701881

18. Suo, Z., Fang, C., Crawford, F., and Mullan. M. (1997) Super-oxide free radical and inter-cellular calcium mediate AP142induced endothelial toxicity. Brian Res. 762, 144152

19. Seilheimer, B., Bohrmann, B., Bondolfi, L., Muller, F., Stuber,D., and Dobeli, H. (1997) The toxicity of the Alzheimers-amyloid peptide correlates with a distinct fiber morphology. J.Struct. Biol. 119, 5971

20. Geula, C., Wu, C. K., Saroff, D., Lorenzo, A., Yuan, M., andYankner, B. A. (1998) Aging renders the brain vulnerable toamyloid -protein neurotoxicity. Nat. Med. 4, 827883

21. Pallitto, M. M., Ghanta, J., Heinzelam, P., Kiessling, L. L., andMurphy, R. M. (1999) Recognition sequence design for peptidylmodulators of amyloid aggregation and toxicity. Biochemistry38,35703578

22. Arispe, N., Pollard, H. B., and Rojas, E. (1996) Zn2 interactionwith Alzheimer amyloid protein calcium channels. Proc. Natl.Acad. Sci. USA 83, 17101715

23. Kawahara, M., Arispe, N., Kuroda, M., and Rojas, E. (1997) Alzheimers disease amyloid -protein forms Zn2-sensitive,cation-selective channels across excised membrane patchesfrom hypothalamic neurons. Biophys. J. 73, 6775

24. Rhee, S. K., Quist, A. P., and Lal, R. (1998) Amyloid betaprotein(142) forms calcium permeable, Zn2-sensitive chan-nel. J. Biol. Chem. 273, 1337913382



11/11

25. Lin, H., Zhu, Y. J., and Lal, R. (1999) Amyloid beta protein(140) forms calcium permeable, Zn2-sensitive channel in recon-stituted lipid vesicles. Biochemistry 38, 1118911196

26. Hirakura, Y., Lin, M. C., and Kagan, B. L. (1999) Alzheimeramyloid A beta 142 channels: effect of solvent, pH, and Congored. J. Neurosci. Res. 57, 458456

27. Arispe, N., Pollard, H. B., Rojas, E. (1993) Giant multilevelcation channels formed by Alzheimer disease amyloid beta-protein [A beta P-(140)] in bilayer membranes. Proc. Natl.Acad. Sci. USA 90, 1057310577

28. Lal, R., and John, S. A. (1994) Biological application of atomicforce microscopy. Am. J. Physiol. 266, C1C21

29. Barbee, K. A., Mundel, T., Lal, R., and Davies, P. F. (1995)Subcellular distribution of shear stress at the surface of flow-aligned and nonaligned endothelial monolayers. Am. J. Physiol.268, H1765H1772

30. Lal, R., Drake, B., Blumberg, D., Saner, D. R., Hansma, P. K.,and Feinstein, S. C. (1995) Imaging real-neurite outgrowth andcytoskeletal reorganization with an atomic force microscope.Am. J. Physiol. 269, C275C285

31. Ivins, K. J., Ivins, J. K., Sharp, J. P., and Cotman, C. W. (1999)Multiple pathways of apoptosis in PC12 cells J. Biol. Chem. 274,21072112

32. Lockart, B. P., Benicourt, C., Junien, J. L., and Privat, A. (1994)Inhibitors of free radical formation fail to attenuate direct-amyloid2535 peptide-mediated neurotoxicity in rat hippocam-pal cultures J. Neurosci. Res. 39, 494550

33. Lane, N. J., Balbo, A., Fukuma, R., Rapoport, S. I., andGaldzicki, Z. (1998) The ultra-structural effects of -amyloid

peptide on cultured PC12 cells: changes in cytoplasmic andintramembranous features. J. Neurocytol. 27, 707718

34. Shearman, M. S., Ragan, C. I., and Iversen, L. L. (1994)Inhibition of PC12 cell redox activity is a specific, early indicatorof the mechanism of beta-amyloid-mediated cell death. Proc.Natl. Acad. Sci. USA 91, 14701474

35. Liu, Y., and Schubert, D. (1998) Steroid hormones blockamyloid fibril-induced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT) formazan exocytosis: relation-ship to neurotoxicity. J. Neurochem. 71, 23222329

36. Zhu, Y. J., Lin, H., and Lal, R. (2000) Fresh and non-fibrillarAmyloid Protein (1-40) induces rapid cellular degeneration inaged human fibroblasts: Evidence for AP-channel mediatedcellular toxicity. FASEB J. 14, 12441254

37. Maestre, G. E., Tate, B. E., Mojacha, R. E., and Marotta, C. A.(1993) Membrane surface ruffling in cells that overexpress Alzhei-

mer amyloid /A4 C-terminal peptide. Brain Res. 621, 14514938. Yankner, B. A., Duffy, L. K., and Kirschner, D. A. (1990)Neurotrophic and neurotoxic effects of amyloid protein:reversal by tachykinin neuropeptides. Science 250, 279282

39. Kosik, K. S., and Coleman, P. (1992) Is -amyloid neurotoxic?Neurobiol. Aging 13, 535630

40. Etcheberrigaray, R., Ito, E., Kim, C. S., and Alkon, D. L. (1994)Soluble -amyloid induction of Alzheimers phenotype forhuman fibroblast K channels. Science 264, 276279

41. Ito, E., Oka, K., Etcheberrigaray, R., Nelson, T. J., Mcphie, D. L.,Tofel-Grehl, B., Gibson, G. L., and Alkon, D. L. (1994) Internal

Ca2 mobilization is altered in fibroblasts from patients withAlzheimer disease. Proc. Natl. Acad. Sci. USA 91, 534538

42. Dikalov, S. I., Vitec, M. P., Maples, K. R., and Mason, R. P.(1999) Amyloid beta peptides do not form peptide derived freeradicals spontaneously. J. Biol. Chem. 274, 93929399

43. Benzi, G., and Moretti, A. (1995) Are reactive oxygen speciesinvolved in Alzheimers disease? Neurobiol. Aging 16, 661674

44. Kim, T. W., Pettingell, W. H., Jung, Y. K., Kovacs, D. M., andTanzi, R. E. (1997) Alternative cleavage of Alzheimer-associatedpresenilins during apoptosis by a caspase-3 family protease.Science 277, 373376

45. Koh, J.-Y., Yang, L. L., and Cotman, C. W. (1990) -amyloid

protein increases the vulnerability of cultured cortical neuronsto excitotoxic damage. Brain Res. 533, 315320

46. Li, J., Xu, M., Zhou, H., Ma, H., and Potter, H. (1997) Alzheimers presenilins in the nuclear membrane, interphasekinetochores, and centrosomes suggest a role in chromosomesegregation. Cell 90, 917927

47. White, A. R., Zheng, H., Galatis, D., Maher, F., Hesse, L.,Multhaup, G., Beyreuther, K. Masters, C. L., and Cappai, R.(1998) Survival of cultured neurons from amyloid precursorprotein knock-out mice against Alzheimers amyloid-beta toxic-ity and oxidative stress. J. Neurosci. 18, 62076217

48. Mattson, M. P., Cheng, B., Davis, B., Bryant, K., Lieberburg, I.,and Rydel, R. E. (1992) -Amyloid peptides destabilize calciumhomeostasis and render human cortical neurons vulnerable toexcitotoxicity. J. Neurosci. 12, 376389

49. Good, T. A., Smith, D. O., and Murphy, R. M. (1996) -amyloid

peptide blocks the fast inactivating K

current in rat hippocam-pal neurons. Biophys. J. 70, 29630450. Sanderson, K. L., Butler, L., and Ingram, V. M. (1997) Aggre-

gates of a beta-amyloid peptide are required to induce calciumcurrents in neuron-like human teratocarcinoma cells: relationto Alzheimers disease. Brain Res. 744, 714

51. Whitson, J. S., and Appel, S. H. (1995) Neurotoxicity of amyloidbeta protein is not altered by calcium channel blockade. Neuro-biol. Aging 16, 510

52. Jansen, J., Ashley, R. H., Harrison. D., McIntyre, S., and Butler,P. C. (1999) The mechanism of islet amyloid polypeptidetoxicity is membrane disruption by intermediate-sized toxicamyloid particles. Diabetes 48, 491498

53. Hirakura, Y., and Kagan, B. L. (1999) Channel formation byserum amyloid A: a mechanism for amyloid pathogenesis andhost defence. Biophys. J. 76, A443 (abstr.)

54. Lin, M., Mirzabekov, T., and Kagan, B. L. (1997) Channelformation by a neurotoxic fragment of the prion protein J. Biol.Chem. 272, 4447

55. Mirzabekov, T., Lin, M., and Kagan, B. L. (1996) Pore formationby the cytotoxic islet amyloid peptide amylin. J. Biol. Chem. 271,19881992

56. Berridge, M. J. (1997) Elementary and global aspects of calciumsignalling. J. Physiol. 499, 291306

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

1243AP142


bathia 2000 toxicity 1.42

Documents