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Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, California 93106, USA
3Correspondence: Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA 93106. E-mail: rlal{at}physics.ucsb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: atomic force microscope • scanning probe microscopy • amyloid beta protein • ion channels • Alzheimer’s disease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
A majority of AD is sporadic (SAD), or nonfamilial in origin, although
several genetic linkages have also been identified (2
3
4
5
6)
.
AßP1–40 is the most prevalent form present in
brain, blood vessels, and other tissues derived from SAD and
normal-aged donors (7
8
9)
. Recent evidence suggests that
AD might be a systemic disease affecting multiple tissues, and AßP
toxicity has been observed in neurons and non-neuronal cells
(10
11
12
13
; for review, see ref 14
).
Significantly, the presence of plaques and comparable toxicity in
non-neuronal tissues provides a window to examine many hypotheses
underlying AßP-induced pathophysiology by using cells derived from
simpler and accessible peripheral tissues, especially aged
tissues; e.g., fibroblasts derived from aged, non-AD humans, releasing
little, if any, soluble AßPs. For a detailed rationale for using
cells from peripheral tissues for such a study, see ref
14
.
The in vitro AßP toxicity has been examined, primarily
after a prolonged incubation (for many hours or days) with low
concentrations (~1 µM) or after short-term treatment (4–6 h) with
relatively higher concentrations (20–50 µM) of
AßP1–40 (10
11
12
13)
. The basic
assumption underlying such studies is that fibrillar plaques are
important for cellular toxicity. Based on such studies, major
prevailing hypotheses underlying the mechanism of AßP toxicity are
included by 1) its interaction with the tachykinin receptors
(15)
; 2) changing cellular ionic level via
existing cation channels (16
, 17)
or via formation of
cation-permeable channels and allowing Ca2+
uptake from an external source (18
19
20
21
22
23
24)
and/or release of
intracellular-stored Ca2+ (25)
; and
3) activating the oxidative pathways (for reviews, see refs
26
, 27
). Reactive oxygen species and the antioxidant
defenses may work by altering lipid peroxidation and membrane
composition (28)
or, indirectly, by altering calcium
uptake via ion channels. Studies examining this mechanism, however,
have produced mixed results on cytoskeletal organization and cell
lysis (13
, 29
30
31
32
33
34)
.
Altered cellular properties and degeneration after a prolonged
incubation of AßPs may reflect a cascade of cellular responses,
including new protein synthesis, altered gene expression, and aging of
the freshly released AßPs. Although the AßP toxicity appears to
correlate with its age and its fibrillar morphology, human AßP
transgenic mice develop fibrillar plaques but do not show comparable
degenerations (35)
. Moreover, several recent studies
suggest that fibrillar form may not be toxic but could be
cytoprotective (36)
.
Globular and nonfibrillar AßP1–40, which is
released continually during normal cellular metabolism, is also present
in AD tissues. They elicit Ca2+ and/or other
cationic currents and form Ca2+-selective
channels in reconstituted membrane, isolated plasma membrane, and the
whole cell (16
17
18
19
20
21
22
23)
. They also allow calcium uptake in
reconstituted vesicles and can alter cellular calcium level (17
, 20
, 21
, 24)
. Cellular responses mediated by these fresh globular
proteins 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 a result 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 refs
37
, 38
) integrated with a light fluorescence microscope
(LFM) (39)
to examine the short-term toxicity of the most
common AßPs—AßP1–40—on human AD-free adult
fibroblasts. A detailed rationale for using fibroblasts as a model
system is explained by Gasparini et al. (14)
Cytoskeletal
ultrastructure and morphology of fibroblasts treated with fresh and
globular AßP1–40 were altered significantly in
the presence of extracellular calcium. These changes were blocked by
anti-AßP1–40 antibody and by AßP-channel
blockers, Zn2+ and Tris. Fluorescently labeled
anti-AßP antibodies were localized in intact cell plasma membrane,
and AßP induced a significant increase of the intracellular calcium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Briefly, cells were grown in DMEM,
supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan,
Utah), 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml
amphotericin (Life Technologies, Paisley, U.K.). Cells were maintained
at 37°C and at a 5% CO2.
Chemicals and AßP(1–40) treatment
Pure AßP1–40 was obtained from Bachem
(Torrance, Calif.). AßP1–40 stock solution
(0.5 mg/ml) was prepared by dissolving the peptides in deionized water
and stored as aliquots at -20°C until used. No DMSO or other
solvents were used. AßPs were sonicated to disperse any fibers before
adding on-line to the cells in culture or adsorbing on glass substrate
for imaging single AßPs. ZnCl2, Tris,
CdCl2, Physalaemin, Trolox, and dithiothreitol
(DTT) were purchased from SIGMA-Aldrich Chemicals (St. Louis, Mo.).
Immunolabeling with anti-AßP antibody
A monoclonal anti-AßP antibody (3D6 concentration=3.75 mg/ml)
against an epitope to the amino terminus (site 2–7) was a generous
gift from Athena Neurosciences, Inc. (San Francisco, Calif.). Goat
antimouse-IgG conjugated with fluorescein (FITC) (1 mg/ml) was
purchased from Chemicon International, Inc. (El Segundo, Calif.).
Cells grown on glass coverslips were incubated with or without AßP (~100 nM concentration) overnight. Immunolabeling was examined in unfixed and fixed cells. For fixation, cells were incubated with 4% paraformaldehyde for 15 min. Cells were washed with PBS and then with PBS containing 3% BSA and 1% goat serum to minimize any nonspecific binding, followed by incubation with 1 µg/ml primary antibody 3D6 for 1 h. After washing, the sample was incubated for 1 h with fluorescein-conjugated secondary antibody. Fluorescence images were captured using a 40x-high NA objective lens with an Olympus optical microscope and/or a Zeiss inverted microscope integrated with the AFM.
Atomic force microscopy
AßP1–40 molecules were imaged with a
Nanoscope III Multimode atomic force microscope (Digital Instruments,
Santa Barbara, Calif.). All samples were imaged in tapping mode using
either a J or a D scanner and silicon nitride tips with nominal spring
constant ~0.32 N/m. The driving frequency and amplitude were chosen
as 8–9 kHz and 10–20 nm. The scan rates were set between 2 and 7 Hz,
and the proportional and integral gains were set between 0.5 and 2.
AßP1–40 was first dissolved in deionized water
and then diluted in PBS to 0.5–2 mg/ml. After incubating in room
temperature between 0 and 48 h, AßP1–40
was deposited on freshly cleaved mica for 10–20 min and imaged under
PBS after a thorough wash to remove unattached molecules.
Fibroblasts were imaged on days 2 and 3 after plating in glass or
plastic petri dishes, using a prototype Bioscope integrated with a
fluorescence microscope (Digital Instruments) in contact and tapping
modes using oxide-sharpened silicon nitride tips (attached to 200 µm
narrow SiN3 cantilever with a nominal spring constant of 0.06 N/m,
Digital Instruments) as described (24)
. Briefly, cells
were imaged in fresh DMEM without any antibiotics but with 20 mM HEPES
or in HEPES-buffered OPTI-MEM reduced serum medium (Life Technologies).
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.
While imaging AßP effects in the absence of extracellular calcium,
gap junctional channel blockers, oleamide and ßGCA, were added in the
medium to prevent a hemi-gap junctional channel opening, which could
occur in the absence of extracellular calcium. After careful optical
alignment, the tip was lowered manually (under visual control with the
light microscope) to 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 adjusted again to the minimum, and the scan size was
gradually increased. The imaging force was regularly monitored and
usually kept to a < nN level. The scan rate varied from 0.1–5 Hz
(scan size 512x512 pixels). All imaging was performed at room
temperature (22–24°C).
The majority of AßP-induced cellular changes were observed within 10–15 min after the addition of AßP1–40 and were pronounced enough in the first 35–45 min after the addition of AßP (time zero). Accordingly, only AFM images at 0 and 45 min are presented, unless otherwise indicated. Cells without AßP treatment (control experiments) were imaged as long as they retained their viability, often for 5 h. Images were captured continually after completion of each scan frame, usually every 3–5 min.
The effect of each perturbation (e.g., antibody, cations, tachykinins) was imaged in separate petri dishes. First, AFM images were captured before treatment with any perturbation, then a perturbation was added on-line, and then again images were obtained continually for up to 2–3 h or up to the time the cells remained viable. For each perturbation, the repeatability of the effect was imaged in at least 6–8 cell clusters/petri dishes.
Submicron-level structural changes are more easily observed at cellular
edges and in the areas of cellular contacts (24
, 40)
.
Also, the size of fibroblasts in our study was often larger than the
AFM scan size limit. Hence, for presentation in this manuscript, we
selected primarily cellular edges, although the structural changes were
examined over the whole cell when possible.
Cell calcium imaging
Intracellular calcium was imaged using a calcium-sensitive dye,
Calcium-Green-AM (Molecular Probes, Eugene, Ore.), and a Bio-Rad MRC
1024 laser confocal microscope, as described (24)
. Cells
were cultured on glass coverslips (22 mm diameter, Fisher Scientific
Co., Pittsburgh, Penn.) coated with collagen IV. To load dye into the
cells, 5 µM Calcium-Green-AM was incubated with cells for 1 h 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. The images were obtained at room temperature and in
HEPES-buffered OPTI-MEM reduced serum medium (Life Technologies); for
the calcium-free condition, a specially ordered nominal calcium-free
HEPES-buffered OPTI-MEM reduced serum medium (Life Technologies) was
used. The excitation and emission wavelengths were selected at 488 nm
and 522 nm, respectively. The objective used for the experiments was a
60x Nikon PlanApo oil emersion lens with a numerical aperture of 1.4.
The focal planes were set across the middle of cell bodies. Images were
collected at 5 sec intervals. The intracellular
Ca2+ concentration was not calibrated for the
present study.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The apparent diameters of most globular
AßP1–40 ranged between 7 and 12 nm. After
48 h of incubation at room temperature (concentration 2 mg/ml),
fibrillar and a large percentage of globular conformations were present
(Fig. 1B
; ref 21
). During the first few hours,
the time period during which we examined
AßP1–40-induced cellular structural changes,
no fibrillar aggregates were observed.
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AßP1–40-induced cellular degeneration
Cytoskeletal organization and cellular processes were imaged in
real time, and we examined the short-term effects of
AßP1–40 treatment. Cellular morphological
changes, including somal shrinkage, plasma membrane blebbing, and
membrane rupture (as viewed in light microscopic and Electron
microscopy images), are commonly used as indicators for cellular
degeneration. We used AFM, which allows imaging cellular morphological
changes on a submicron scale that could result during a short-term
AßP1–40 treatment. Because submicron-level
structural changes are more easily observed at cellular edges and in
the areas of cellular contacts, and because the cell size was often
larger than the AFM scan size, we selected only the cellular edges for
representation, although both large and small cells were imaged.
In cells without AßP treatment, no substantial cytoskeletal
reorganization was observed, and cells maintained stable morphology
over continual imaging even after 5 h (Fig. 2A, B
). On-line addition of 1 µM AßP1–40 induced a
significant change in the cell morphology, which began quickly (within
10–15 min of incubation) and continued with a gradual but irreversible
loss of cell morphology. Figure 2C, D
shows the disruption
of cell–cell connections and the retraction of cellular processes
within 30–40 min. The short-term changes in central regions of
AßP1–40-treated cells were less pronounced
compared with the peripheries. However, by reducing the imaging force,
it was possible to image only the cell surface structures, and in
the presence of AßP1–40, the cell surface
appeared significantly more convoluted and tangled in comparison with
the control, non-AßP-treated cells’ plasma membrane. 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 ( 24
; for review, see
ref 37
). In the present study, the degenerative
morphological changes were apparent only in the
AßP1–40-treated and not in the untreated
cells. Also, no difference in AßP1–40 effect
was observed for cells incubated in DMEM (plus 20 mM HEPES) or
HEPES-buffered OPTI-MEM reduced serum medium.
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Based on our preliminary investigations, the rate of loss of cellular processes appears to be dependent on the concentration of AßP1–40, and the cellular degeneration is accelerated in the presence of a higher level of AßP1–40. The rate of cellular degeneration could also depend on other factors like the cell size, cell cycle stage, cell density, level of cellular communication, and temperature. In our study, the concentration of AßP1–40, which could induce cellular degeneration, was as low as 200 nM. A quantitative relationship between the extent of cellular degeneration and toxicity and the concentration of AßP1–40 is currently under investigation.
The repeatability of AßP1–40-induced cellular changes was observed in five to eight other cell clusters in the same petri dish and in six additional sets of experiments. AßP1–40 induced cellular degeneration in most of the cells (>85–90%). In addition to higher resolution AFM imaging, we also captured light video microscopy images with the integrated light microscope (20x obj). This allowed us to examine the overall AßP1–40-induced changes in the morphology of populations of cells in a petri dish. In parallel with the AFM study of single cells, there was an overall degeneration of the majority of cells in AßP1–40-treated petri dishes.
Significantly, in comparison, AßP1–40 did not
induce any measurable cellular degeneration in cultured endothelial
cells when applied at a similar concentration (24)
.
Endothelial cells, however, showed significant degeneration in response
to treatments with nanomolar AßP1–42
(24)
. AßP1–40 is also reported to
induce time- and concentration-dependent ultrastructural changes in
PC12 cell membranes (41)
.
Such AßP1–40-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, which 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
(42
, 43)
. Furthermore, it is unclear if AßP-induced cell
death is an apoptotic or a necrotic process.
Specificity of AßP toxicity
When an equal molar concentration of anti-AßP antibody was
added, 1 µM AßP-induced cellular damage was still present
(Fig. 3A, B
). However, for 10x antibody (membranes bound and free in the media to
bind to most of the AßP molecules), the AßP toxicity was nearly
completely prevented: cell–cell connection was preserved, and cells
retained their normal morphology (Fig. 3C, D
). The plasma
membrane, however, became rougher, perhaps representing the
membrane-bound antibodies.
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Figure 4
shows immunofluorescence labeling of fibroblasts using 3D6 anti-AßP
antibody. Immunolabeling is present across the whole cell surface of
cells incubated with AßP (Fig. 4A
). For comparison, cells
not incubated with AßP show no immunolabeling (Fig. 4C
).
Corresponding phase contrast images are shown in Fig. 4B, D
.
A simple comparison of panels Fig. 4A, B
and Fig. 4C, D
suggests that the immunolabeling is limited to cell boundary
only, and nonspecific binding to the substrate, if any, is minimal.
Immunolabeling was observed in unfixed and fixed cells, although the
labeling was stronger for the unfixed cells. In AßP-treated cells, a
stronger labeling was observed above the nuclear region and along the
cell boundaries (Fig. 4A
), perhaps reflecting a larger
surface area resulting from the AßP-induced plasma membrane extension
and ruffling (44
, 45)
or a preferential regional
incorporation of AßP in the plasma membrane.
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Mechanism of AßP action
Several mechanisms of AßP toxicity have been postulated and
include: 1) AßP-induced enhanced responsiveness to
oxidative stress (for review, see refs 26
, 27
);
2) the interaction of AßP with tachykinin receptors
(15)
; or 3) the AßP-mediated modulation of
cation-selective, pre-existing, or newly formed ion channels
(16
17
18
19
20
21
22
23
24
25)
.
When physalaemin, a tachykinin, was added before or along with
AßP to prevent binding of AßP to potential tachykinin receptors, a
significant change in the cytoskeletal network with the eventual loss
of cell–cell contacts was still observed (Fig. 5B
). Such cellular degeneration was observed over a range of physalaemin
concentration tested. These results suggest that
AßP1–40 toxicity is not mediated via its
binding to tachykinin receptors. In our preliminary study, antioxidants
Trolox and DTT were not able to prevent
AßP1–40 toxicity. In contrast, on-line
addition of zinc or Tris significantly inhibited
AßP1–40-induced cellular degeneration over the
similar time period (Fig. 5D, F
, respectively). Such
protection from AßP toxicity was observed when zinc/Tris were added
before, along with, or shortly after the addition of
AßP1–40.
|
Significantly, when the extracellular medium was devoid of
calcium, AßP1–40 incubation did not induce any
visible, cellular, morphological changes or degeneration. Figure 6
shows example of a lack of any significant structural changes in 45 min
of AßP1–40 treatment when the cells were
incubated in no-calcium medium (compare Fig. 6A, B
). After returning the extracellular calcium to normal
level, however, the cellular degeneration resumed. These results
suggest that AßP1–40 toxicity is mediated via
intracellular uptake of extracellular calcium via zinc-sensitive,
calcium-uptake mechanism.
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Imaging intracellular calcium
To ascertain whether there is indeed an AßP-induced increase in
the intracellular calcium level, we imaged AßP-induced change in the
cytoplasmic calcium level using a calcium-sensitive dye, Calcium-Green,
and laser scanning confocal microscopy. Figure 7A
shows the resting intracellular calcium levels before
AßP1–40 was added.
AßP1–40 (1 µM) was then added to the bathing
solution (HEPES-buffered OPTI-MEM). The intracellular calcium level
increased significantly within minutes. The peak of intracellular
Ca2+ level was reached 10–12 min after AßP
addition (Fig. 7C
). The fluorescence intensity decreased
gradually after 12–15 min (Fig. 7D, E, F
) but
remained at an elevated level compared with the pre-AßP state even
after 30 min (Fig. 7E
). The AßP-induced increase is
dependent on the presence of extracellular Ca2+.
In the absence of extracellular Ca2+, no
significant Ca2+ increase was observed even when
cells were incubated with a relatively high concentration (4.4 µM) of
AßP1–40 (Fig. 8
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fresh and globular AßP1–40 treatment induced rapid
cellular degeneration
The effect of AßP treatment on cellular degeneration is rapid
and specific. AßP induces changes in the morphology of the cell
surface and the cytoskeleton. AFM images show that AßP altered the
cytoskeleton dramatically within 15–20 min. Moreover, in AßP-treated
cells, cell-surface ruffling was also apparent. AßP-induced
plasma-membrane ruffling has been reported in PC12 cells
(45)
and several other modifications, including the
presence of intramembranous particles (IMPs), are reported in
AßP-treated cells (41)
. It is unlikely that these
cellular modifications observed in our study are affected by the
imaging artifacts, specifically the imaging force, because the imaging
force can be controlled quite well for a nonperturbed imaging of
cellular structures, including cell surface and the cytoskeletal
network (24
, 40)
(for reviews, see refs
37
38
39
). Moreover, in the present study the cellular
modifications were present only in the AßP-treated cells and were not
present in the non-AßP-treated cells. Similar results were obtained
in AßP1–42-treated endothelial cells using
identical experimental paradigms (24)
.
AFM images of AßP1–40 prepared in PBS, from
the stocks dissolved in deionized water, show that they retain globular
conformations for an extended time period. Further, they did not form
fibrillar structure, even at a much higher concentration, over the time
period during which cellular degeneration was examined. Previous
studies have shown that they form fibers when aged for an extended time
period. Such a difference in the conformations of
AßP1–40 could be because of several factors,
including the choice of solvent for making the stock solution and the
final solution, the purity of AßPs, and the source of the peptide.
Significantly, we have shown recently that the AßP used in our study
allows Ca2+ uptake when reconstituted in lipid vesicles
(20
, 21)
, and forms a pore-like structure (preliminary
results; ref 46
).
Specificity of AßP toxicity
AßP-induced cellular degeneration was blocked in the presence of
anti-AßP antibody and thus provides strong evidence that the
degeneration is specific to AßP1–40 treatment.
It also argues against any possible imaging artifact, including tip- or
imaging force-induced damages. Although not tested in the present
study, scrambled AßPs (e.g., AßP40–1) should
not produce any cellular degeneration, because they do not show
cation-selective channel activity when reconstituted in artificial
membranes (B. Kagan, personal communication) and also do not allow
calcium uptake (unpublished observation). Consistent with the specific
AßP1–40 toxicity to fibroblasts,
AßP1–40 did not induce any significant
degeneration in endothelial cells in an otherwise similar experimental
condition (24)
.
As further evidence for the AßP1–40-specific
toxicity, AßP1–40 was immunolocalized in the
plasma membrane of live fibroblasts without any fixation. Fibroblasts
not treated with AßP1–40 showed no
immunolabeling. Because the antibody used in our study is specific to
the amino terminus portion of AßP, these results suggest that added
AßP1–40 were incorporated into the cell plasma
membrane with the NH2 terminus of the peptide
located outside of the membrane. Significantly, 3-D models of
AßP-channel structure support such binding epitopes of site-specific
antibodies and ligands (47)
. Many cell types, including
fibroblasts, express endogenous acute-phase proteins (APP) and secrete
soluble AßP (48
, 49)
. A lack of immunolabeling in
fibroblasts not incubated with AßP1–40
(control) in our study could be because of several reasons. First,
fibroblasts derived from aged non-AD humans release very little, if
any, soluble AßPs (49)
. Second, in our study, before
incubation with exogenous AßPs, cells were extensively rinsed to
remove endogenously released AßP. Third, the conformation of the
endogenous APPs may be such that the antibody recognition site is
inaccessible for antibody binding.
Mechanism of AßP toxicity
AßP-induced cellular degeneration was not blocked by physalaemin
over a wide range of concentrations tested, when applied with or before
AßP1–40 treatment. On the contrary,
physalaemin accelerated AßP1–40-induced
cellular degeneration, although prolonged incubation of physalaemin
alone did not cause any significant change in cell morphology. Thus,
the AßP1–40 toxicity in cultured fibroblasts
does not appear to be modulated by its binding to tachykinin-like
neuropeptide receptors and activating secondary signal transduction
pathways. Physalaemin and other tachykinins were reported to prevent
AßP toxicity in neuronal cells (13)
. Such difference in
the action of tachykinins may reflect the effect to be cell-type
dependent (neuronal vs. non-neuronal). However, AßP toxicity has been
shown to be comparable and similar in neuronal and non-neuronal cells
and a result of a lack of a sufficient level of tachykinin receptors in
the fibroblasts used in our study.
AßP toxicity may also occur by AßP-induced and increased
sensitivity to free radicals. In our experiments, we used freshly
thawed AßPs—fresh proteins presumably contain none to only a trace
amount of free radicals—and the toxicity began immediately after the
AßP addition. Garzon-Rodriguez et al. (50)
have shown
that AßP1–40 exists as stable molecules very
low (nanomolar) to the critical concentration (~25 µM). The stable
molecules do not appear to form fibrils and remain globular over an
extended time period (Fig. 1)
(21
, 51)
. This is consistent
with our observation that the AßP toxicity in these fibroblasts is
probably not mediated by the formation of free radicals. Moreover, we
have just recently shown that antioxidants do not prevent toxicity of
more active AßP1–42 in endothelial cells
(24)
.
Our results strongly suggest that the AßP toxicity is mediated
directly via Ca2+-permeable pores made from fresh
AßPs. Consistent with such assertion that 1) in the
Ca2+-free medium, AßP incubation did not induce
any cellular degeneration, and 2) AßP-induced cellular
degeneration was blocked by zinc and Tris, which were previously shown
to modulate cation conductances in AßP-reconstituted vesicles and in
various cell types (18
, 19
, 22
, 52)
. Previously,
AßP1–40 was shown to elicit cation-selective
ionic currents (primarily K+ current) in
fibroblasts used in the present study (16
, 17)
. Thus, it
is likely that AßP1–40 acts via existing ion
channels and by making its own channels. A presence of AßP in the
whole cell plasma membrane (Fig. 4
and ref 41
) will be
consistent with the possibility of AßP1–40
forming cation-selective channels i the plasma membranes.
An altered cation homeostasis underlies AßP-toxicity (16
, 17
, 24
, 25)
. We have shown a significant and sustained increase in
the intracellular Ca2+ level in cells treated
with AßP1–40 (Fig. 7)
but not in the absence
of extracellular calcium (Fig. 8)
. An AßP-induced, sustained,
elevated Ca2+ level is, most likely, a result of
Ca2+ uptake from the external medium via AßP
pores (46)
, and AßP channel-mediated calcium uptake was
reported for other cell types (24
, 53)
. Elevated calcium
could also result from the binding of AßP to cell membrane receptors,
thus acting as a signaling stimulus for activating tyrosine and Ser/Thr
phosphorylation, and induction of transcriptions. Our limited study
could not resolve whether the calcium increase is indirectly via AßP
cell membrane-receptor interactions or directly via
Ca2+-permeable AßP channels, or both. The
direct pathway via Ca2+-permeable AßP channels,
however, appears to be the most likely mechanism, based on studies
showing 45Ca2+ uptake in AßP-vesicles
(20
21)
and Ca2+-sensitive currents
in AßP1–40-reconstituted lipid membranes
(18
, 19
, 22
, 23)
. An elevated calcium level alters
micromechanical properties of virtually all cell types
(54)
and induces significant structural reorganization. It
is quite likely that an AßP1–40-induced
increase in calcium level leads to a toxic level, which gradually
induces irreversible cytoskeletal reorganization. Indeed, cytoskeletal
disruption is one of the earliest detectable changes correlating with
neurodegenerative disorders such as AD (55)
.
In summary, we show that fresh and nonfibrillar
AßP1–40 induces rapid cellular reorganization,
including loss of cytoskeletal network, cell–cell connections, and the
retraction of cellular processes. Such cellar degeneration is mediated
by elevating the level of intracellular calcium, most likely through
cation-permeable AßP channels (21)
and not by its
interaction with the tachykinin receptors or by AßP-induced and
enhanced responsiveness to free radicals. The short exposure, high-dose
toxicity is predictive of the low-dose, long exposure toxicity as in
other diseases, such as cancer and heart disease. (A low level of free
AßPs forms relatively fewer AßP channels and allows a low level of
Ca2+ uptake, which can be managed by a healthy
cell.) However, a continued accumulation of AßP channels over an
extended time period can allow a pathologically high level of
Ca2+ uptake leading to cell toxicity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Molecular Imaging Corp., Phoenix, AZ 85044, USA.
Received for publication October 21, 1999. Revised for publication February 3, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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