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* Department of Pharmacology, College of Medicine and Department of Molecular Pharmacology, Neuroscience Research Institute, MRC Seoul National University, Biomedical Brain Research Center, NIH, Seoul 110–799, South Korea;
Department of Pharmacology and Toxicology, Kyorin University, School of Medicine, Mitaka, Tokyo 181, Japan; and
§ Department of Chemistry, College of Natural Science, Konkuk University, Seoul, Korea
1Correspondence: Department of Pharmacology, College of Medicine, Seoul National University, Yongon-dong, 28, Chongno-ku, Seoul 110–799, Korea. E-mail:yhsuh{at}plaza.snu.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: amyloid precursor protein • Intracellular free calcium concentration • Alzheimer’s disease • glutamate • ß-sheet conformation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Although amyloid beta peptide (Aß), a 39 to 43
amino acid-long and principal constituent of senile plaques
(2)
, is the most extensively studied toxic fragment of an
integral membrane protein termed amyloid precursor protein (APP)
(3)
, other proteolytic products of APP may also contribute
to the pathogenesis of AD.
To date, at least three processing pathways of APP have been described:
the nonamyloidogenic secretory pathway (
-secretase), which releases
soluble ectodomain and prevents Aß formation (4)
; the
endosomal-lysosomal pathway, in which some cell APPs are reinternalized
(5)
and cleaved around at the NH2
terminus of the Aß sequence by ß-secretase to produce Aß-bearing
amyloidogenic breakdown products of various sizes (14–22 kDa)
(5
, 6)
and then possibly cleaved by 
-secretase to
release soluble 4 kDa Aß (7)
; and the 4 kDa
Aß-producing pathway (ß-and -secretases) involving coated
pit-mediated endocytosis, which may be independent of the constitutive
secretory pathway (7)
.
Many studies have shown that Aß is toxic to neurons in
vitro (8
, 9)
and in vivo (10
, 11)
. The toxicity of Aß has been known to be related to some
intrinsic ion channel activity (12)
, reactive oxygen
species (9)
, and excessive Ca2+
influx, altering neurotransmitter receptor function (13)
and disrupting Ca2+ homeostasis (8)
.
However, a relatively high concentration (> 20 µM) of Aß was
needed to exert toxicity, and some studies failed to demonstrate the
toxicity of Aß in vivo (14)
. It has been
reported that under certain conditions in culture, Aß promotes
neurite outgrowth (13)
instead of exerting toxic action.
In addition, there are highly variable numbers of Aß-containing
diffuse plaques in normal elderly people, and Aß deposition has been
observed in various brain areas without accompanying neurodegeneration
(15)
. Thus, Aß may not be the sole fragment in the
neurotoxicity associated with AD. Consequently, the possible effects of
other cleavage products of APP need to be explored.
It was reported that carboxyl-terminal fragments of APP (CTs) are found
in media and cytosol of lymphoblastoid cells obtained from patients
with early- or late-onset familial AD (16)
and Down’s
syndrome (17)
. In addition, the carboxyl-terminal peptides
have been identified in plaques, microvessels, and the neurofibrillary
tangles in the brains of AD patients (18
, 19)
. These
carboxyl-terminal fragments, which contain the complete Aß sequence,
appear to be toxic to neurons in culture, although the mechanism of
action is not understood (20
, 21)
. Transgenic mice that
overexpressed the CT 100 peptide (22)
showed extensive
neuronal degeneration in the hippocampal area (23)
and
cognitive impairment (24)
. Recent work has reported that
APP mutations that cause familial Alzheimer’s disease increase the
intracellular accumulation of potentially amyloidogenic and neurotoxic
carboxyl-terminal fragments of APP in neurons (25)
. This
indicates that the carboxyl-terminal fragment of APP might be
significantly involved in the pathogenesis in AD.
Because CT has been presumed to exert its toxic action through secondary generation of Aß, the role of CT peptide itself in the pathogenesis of AD is poorly understood.
We previously reported the neurotoxicity of a carboxy-terminal fragment
of APP that may be released into the extracellular space (26
, 27)
and also shown that CT 105, a recombinant carboxyl-terminal
105 amino acid fragment of APP, induced strong nonselective inward
currents in Xenopus oocytes (28)
, Purkinje
cells (29)
, planar lipid bilayer (30)
as well
as memory deficits and neuropathological changes, characteristics of AD
in mice (31)
.
The secondary structure determines several important properties
of peptides that may be relevant to the pathogenesis of
neurodegenerative diseases. In the case of Aß, it has been
demonstrated that the amyloid peptide is neurotoxic (9
, 13
, 32)
and that this characteristic is associated with the
formation of ß-sheet or amyloid fibrils (33
, 34)
. The
ability of Aß to form fibrils is directly correlated with the content
of ß-sheet structures adopted by the peptide (35)
.
Polymerization of Aß into protease-resistant fibrils is a significant
step in the pathogenesis of AD (36)
. CT is also reported
to able to form self aggregates in vitro (26
, 37)
and in transfected cells (20
, 38)
. The main
determinant of amyloid formation is the conformation adopted by the
peptide in the stage before aggregation (39)
.
In this study, we have investigated the effects of CT 105 on calcium homeostasis and glutamate-induced excitotoxicity in various cultured cells. We performed conformational studies of CT 105 using a circular dichroism (CD) technique to investigate the structural basis for CT aggregation and toxicity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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35 dishes coated with polyethylene (0.2 mg/ml in
sodium borate buffer, pH 8.3) at a density of 2 x
105 cells per dish. After overnight incubation in
Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Inc.,
Grand Island, N.Y.) supplemented with 10% fetal bovine serum, the
medium was changed to serum-free defined medium for neurons [DMEM
supplemented with 2 mM glutamine, 1 mM pyruvate,
penicillin-streptomycin-amphotericin B mixture (Life Technologies,
Inc.), 5 mM HEPES, 0.5% glucose, 10 µg/ml insulin, 30 nM sodium
selenite, 20 nM progesterone, 100 µM putrescine, and 20 ng/ml
transferrin]. The cultures were incubated at 37°C in 5%
CO2 and the medium was replaced every other day.
Experiments were performed in 14- to 15-day-old cultures.
SK-N-SH cells originating from human neuroblastoma were purchased from
Cell Bank of Seoul National University; PC 12 cells originating from
rat pheochromocytoma (40)
were obtained from Dr. Myung
Goo-Lee; and U251 cells derived from human glioblastoma
(41)
were provided by Dr. Y. Sakaki. Cells were plated in
polyethylene-coated coverslips and 96-well plates.
Preparation of recombinant CT 105
Recombinant CT 105 was prepared on the basis of human APP 770
cDNA as described previously (42)
. Briefly, the expression
plasmid pCS-CT 105 was constructed by ligating the 704 bp
BglII-ClaI fragment excised from pSP65-APP 770
into ptrpSF9 (digested with BglII, SmaI and
treated with CIAP) and transformed into Escherichia coli,
XL1-Blue. CT 105 peptide (Mr 14,242)
was purified by a combination of urea solubilization and ion exchange
chromatography and then subjected to dialysis against 10 mM Tris-HCl
(pH 7.4), followed by lyophilization.
Determination of intracellular calcium concentration
The methods for the
[Ca2+]i measurements have
been described (43)
. Briefly, cultured cells were washed
twice with Hank’s solution and incubated at 37°C for 45 min in
Hank’s solution containing 10 µM Fura-2/AM, 5 mM glucose, and 1 mM
CaCl2. To remove the extracellular Fura-2/AM, the
loaded cells were rinsed three times with the same solution. The cells
were placed in a two wavelength microscopic fluorometer (InCytIm2;
Intracellular Imaging Inc., Cincinnati, Ohio). Excitation wavelengths
of 340 and 380 nm and an emission wavelength of 510 nm were used for
the Fura-2 fluorescence. In all experiments, autofluorescence at both
340 and 380 nm wavelengths was extracted from the total fluorescence
intensity increased by treated substances. The fluorescence ratio was
obtained by dividing the fluorescence excited at 340 nm by that 380 nm,
and the [Ca2+]i was
calculated based on the formula described by Grynkiewicz et al.
(43)
:
[Ca2+]I =
[(R-Rmin)/(Rmax-R)] x (Fmax/Fmin) x Kd, where Rmin
is the ratio at zero calcium in the medium, and Rmax is the ratio at
saturation calcium concentration. Fmin is the fluorescence at 380 nm at
zero calcium concentration in the medium and Fmax is the fluorescence
at 380 nm at saturation calcium concentration. The effective
dissociation constant (Kd) of 224 nM for the
Fura-2-Ca2+ complex was used as reported by
Grynkiewicz et al. (43)
.
Calcium imaging by confocal laser scanning microscopy
The cells, pretreated with CT 105, were washed twice with
Hank’s solution and incubated for 20 min at 37°C in the same buffer
containing 10 µM Fluo-3/AM. Images were obtained using a
laser-scanning confocal microscope (Bio-Rad MRC 1024) connected to an
inverted microscope (Zeiss, Germany) fitted with a 10 x 63 x oil. Excitation was achieved using an Argon ion laser [wavelength
(
)=488 nm] and fluorescence was measured at >515 nm. Changes of
intracellular calcium levels were measured using relative fluorescence
compared with that of control group.
Toxicity assay
The toxicities of CT 105 or
Aß1–42 or Aß25–35
with or without cholesterol, MK 801, nifedipine, verapamil, and
glutamate were assessed by lactate dehydrogenase (LDH) assay. After the
appropriate incubation time with the peptides with or without the
various agents listed above, LDH activities in the medium were measured
by a Cytotox 96 nonradioactive cytotoxicity assay kit (Promega,
Madison, Wis.) according to the manufacturer’s instructions. The
results were expressed as percentages of peak LDH release obtained on
complete cell lysis.
Circular dichroism studies
Concentration of CT 105 for CD measurement was 0.05 mM. Sodium
dodecyl sulfate, dodecyl phosphocholine (DPC) micelle solutions were
prepared by dissolving appropriate amount of detergent in 50 mM
phosphate buffer at pH 7.0. DPC was purchased from Avanti Polar Lipids
(Birmingham, Ala.). PC and phosphatidylglycerol (PG) from egg yolk were
purchased from Sigma (St. Louis, Mo.). Small unilamellar vesicles (SUV)
of defined lipid composition were prepared by sonication technique
described by Barenholtz et al. (44)
. CT 105 and vesicles
were incubated in 50 mM phosphate buffer (pH 7.0) for 20–24 h at
25°C prior to CD measurement.
CD experiments were carried out on a Jasco J720 spectropolarimeter in
the Korea Basic Science Institute. All measurements were performed at
room temperature using quartz cells with a path length of 0.1 cm. Data
were collected at 0.1 nm intervals and 8 scans were averaged with the
scan rate of 100 nm/min. The spectra were corrected for buffer and
vesicles after acquisition. The secondary contents were determined by
computer fitting to a library of CD spectra using the learning neural
network program K2D, which is based on the algorithm published
previously (45)
.
Statistical analysis
Statistical analysis was performed by Student’s t
test and ANOVA using an SPSS program (SPSS Inc., Chicago, Ill.) to
study the relationship between the different variables. Values of
P<0.05 were considered to indicate statistical
significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.).
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We have also shown the concentration- and time-dependent changes
of calcium levels with 10 µM Fluo-3/AM in SK-N-SH cells by using
confocal laser scanning microscopy. The pretreatment of SK-N-SH cells
with 1 µM, 2.5 µM, 5 µM, 10 µM of CT 105 for 24 h induced
the concentration-dependent increase in relative fluorescence by
Fluo-3/AM as shown in Fig. 2a, c
. Aß1–42 50 µM elicited a
calcium-increasing effect similar to that of 10 µM of CT 105 (Fig. 2a
). Pretreatment of SK-N-SH cells with 10 µM of CT 105
for 1, 3, 6, 24 h induced a time-dependent increase in relative
fluorescence by Fluo-3AM (Fig. 2b
).
|
To determine whether the elevations in intraneuronal calcium levels induced by CT 105 were due to extracellular calcium influx or not, we measured the change of intracellular calcium concentration and cell cytotoxicity using calcium-containing or calcium-deficient media in SK-N-SH cells. Calcium-deficient medium consisted of HBSS containing no added calcium. There was no change in intracellular calcium concentration in calcium-free media, whereas cells in normal media treated with 10 µM CT 105 for 6 h showed significant increase in calcium concentration (data not shown), suggesting that calcium may enter the cells from outside. Also, cells in the calcium-deficient medium were protected against the neurotoxicity of CT 105 (data not shown). Thus, calcium influx should be necessary for the neuronal damage induced by CT 105.
The effects of neuroprotective agents on the increase in
intracellular calcium concentration and cell toxicity induced by CT 105
To investigate the effects of neuroprotective agents on CT
105-induced neurotoxicity and calcium-increasing effect, we measured
cell viability and changes in intracellular calcium concentration after
pretreatment of 500 µM of cholesterol, 100 µM of MK-801 4 h
before CT 105 treatment and 20 µM of nifedipine and 25 µM of
verapamil 2 h before CT treatment in SK-N-SH and PC 12 cells. We
measured cell viability after treating Aß1–42
and Aß25–35 with or without neuroprotective
agents. Cholesterol significantly inhibited the CT 105-induced increase
in intracellular calcium levels and cell toxicity and MK-801 attenuated
intracellular calcium increase and cytotoxicity in SK-N-SH cells
(Fig. 3
, Fig. 4a
); in the case of Aß1–42 and
Aß25–35, nifedipine and verapamil, specific
blockers of voltage-sensitive calcium channels, protected Aß-induced
toxicity in SK-N-SH (Fig. 4c
) and PC 12 cells (data not
shown), respectively. In PC 12 cells, cholesterol and MK 801 also
reduced CT 105-induced cytotoxicity whereas nifedipine and verapamil
did not (Fig. 4b
).
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The effects of CT 105 on the glutamate-induced excitotoxicity
In this study, CT 105 enhanced glutamate-induced excitotoxicity in
SK-N-SH cells and rat primary cortical neurons (Fig. 5
, Fig. 6
). When these cells were exposed to two concentrations of glutamate (100
µM and 1 mM) and/or three concentrations of CT 105 (500 nM, 1 µM, 5
µM) for 24 h, neuronal survival was significantly reduced
compared to cultured cells incubated in the presence of glutamate
alone. With increasing concentrations of CT 105 from 500 nM to 5 µM,
there was a progressive enhancement of glutamate neurotoxicity (Fig. 5)
. Phase-contrast micrographs in Fig. 6
show fields of cultured rat
cortical primary neurons before and after exposure to 1 mM glutamate
alone or with CT 105 1 µM. The glutamate-induced neuronal
degeneration, enhanced by CT 105, was characterized by vacuolation of
the cell bodies, neurite fragmentation, and ultimately cell death (Fig. 6)
.
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Conformational studies of CT 105 by CD spectroscopy
CD spectra of CT 105 in phosphate buffer, aqueous
2,2,2-trifluoroethanol (TFE) solution, and DPC micelle are presented in
Fig. 7a
. Table 1
lists the predicted secondary structure composition according to our CD
data using the K2D learning neural network program. CT 105 adopts a
partial -helical conformation in phosphate buffer and 50%(v/v)
aqueous TFE solution, with a helical content of 34% and 30%,
respectively. In the case of DPC micelle, the content of ß-sheet
conformation of CT 105 in DPC micelle was 19%, 21%, and 23% in 10
mM, 50 mM, and 100 mM DPC micelles, respectively. Addition of NaCl (up
to 100 mM) did not show any significant effects on the conformation of
CT 105. CD spectrums of CT 105 in SUV are shown in Fig. 7b
.
In both vesicles, CT 105 has partial -helical conformation, but in
the presence of a negatively charged vesicle such as PC/PG (3:1)
vesicle, the content of ß-sheet conformation increased from 16% to
22%, as listed in Table 1
. CD results show that the ß-sheet
conformation of CT 105 may cause self-aggregation and cytotoxicity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 9)
and in vivo (10
, 11
, 46)
. To date, most research has been focused on the toxicity of
Aß related to AD. However, as there are still some discrepancies
between the deposition of Aß and neuronal loss in AD, Aß may not be
the sole causative fragment in AD. Recent works have shown that CT
fragments of APP are found in media and cytosol of lymphoblastoid cells
obtained from patients with early- or late-onset familial AD
(16)
and Down’s syndrome (17)
. In addition,
the CT peptide has been identified in plaques and microvessels and may
be a constituent of the neurofibrillary tangles characteristic of AD
(37)
. These CT fragments of APP, which contain the
complete Aß sequence, appear to be toxic to neurons in culture,
although the mechanism of action is not fully understood
(38)
. Furthermore, transgenic mice that overexpressed the
CT 100 peptide (22)
showed extensive neuronal degeneration
in the hippocampal area (23)
as well as cognitive
impairments (24)
and inhibition of long-term potentiation
(LTP; 47
). Because CT has been presumed to exert its toxic
action through secondary generation of Aß, however, the role of CT
peptide itself in the pathogenesis of AD is poorly understood.
Recent work has reported that APP mutations that cause familial
Alzheimer’s disease increase the intracellular accumulation of
potentially amyloidogenic and neurotoxic carboxyl-terminal fragments of
APP in neurons (25)
. These results suggest that the
carboxyl-terminal fragment of APP is significantly involved in the
pathogenesis in AD, especially in familial AD.
We earlier reported that either extracellular or intracellular
application of CT 105 elicited strong nonselective inward currents and
toxic effects in Xenopus oocyte (28)
, rat
Purkinje neuron (29)
, PC 12, and cultured rat cortical
cells (26)
, and that CT might be released into the media
(26)
and may affect surrounding neurons and CT blocked LTP
in rat hippocampus in vivo (48)
and induced
behavioral impairment and neuropathological changes in mice
(31)
.
In the present study, we focused on the effects of CT 105 on the
intracellular calcium homeostasis and glutamate-induced excitotoxicity
in various cultured cells. Alterations in calcium homeostasis can lead
to intracellular calcium accumulation and trigger lethal process
(49)
. We demonstrate that the treatment with 10 µM CT
105 for 24 h increased intracellular calcium concentration in the
SK-N-SH and PC 12 cells by about twofold and not significantly in U251
cell. This result is consistent with our previous results that CT 105
directly damaged the cortical neurons and PC 12 cells but did not
affect the viability of U 251 cells originating from human
glioblastoma, suggesting a neuroselective toxic effect of CT 105
(26)
. It has been reported that neuronal cells were much
more sensitive to the toxic effects of amyloidogenic CT fragments.
Neurons but not non-neuronal cells derived from a multipotent embryonic
carcinoma cell line (P19 cells) and a morphologically heterogeneous
neuroblastoma cell line (SK-N-SH) were vulnerable to overexpression of
CT 99 (19)
. The mechanism of cell type-dependent toxic
effects of CT 105 is not clear. However, it is likely that CT 105
exerted a calcium homeostasis-destabilizing effect only on neuronal
cell and not on non-neuronal cell, owing to the different membrane
structure in neuronal cell and non-neuronal cell. In our results,
cholesterol could significantly attenuate CT 105-induced neurotoxicity
and increase in intracellular calcium levels, whereas the cytotoxicity
induced by Aß1–42 and
Aß25–35 was significantly reduced by
nifedipine and verapamil in SK-N-SH and PC 12 cells. This is consistent
with the previous report by others that the neurotoxicity by
Aß1–40 was attenuated by nimodipine, a
voltage-sensitive calcium channel blocker (50)
. Taken
together, these results can suggest that CT 105 and Aß exert
neurotoxic effects by different mechanisms.
Although the mechanism whereby CTs destabilize calcium homeostasis is not known, several possibilities are worth considering.
CT was reported to form cation-selective ion channels in the
planar lipid bilayer composed of palmitoyloleoylphosphatidylethanolamine and
palmitoyloleoylphosphatidylcholine (80:20) (30)
. The
intracellular calcium-increasing effect of CT 105 shown in this study
might be due to this channel-forming effect of CT 105. This
channel-forming effect could be strongly supported by results by Fraser
et al. (28)
, who reported that CT 105 induced strong
nonselective inward currents in Xenopus oocyte.
In this study, there was no change in intracellular calcium concentration in calcium-free media, whereas cells in normal media treated with 10 µM CT 105 for 6 h showed a significant increase in calcium concentration, suggesting that calcium may enter the cells from outside. Also, cells in the calcium-deficient medium were protected against the neurotoxicity of CT 105. Thus, calcium influx should be necessary for the increase in intracellular calcium and neuronal damage induced by CT 105.
Results showing that cholesterol, an agent that decreases membrane
fluidity, could protect SK-N-SH and PC 12 cells from cell toxicity and
calcium increase by CT 105 could support the hypothesis that CT 105
might increase calcium concentration by a channel-forming effect.
Intracellular injection of CT 105 into Xenopus oocyte caused
the same channel effects but to a greater extent than extracellular
application of CT 105, suggesting that extracellular reflects an
intracellular effect (28)
. In this study, the increase in
intracellular calcium and toxicity exerted by CT 105 in SK-N-SH and PC
12 cells was also attenuated by MK-801, a noncompetitive NMDA receptor
antagonist, which raises the possibility that CT 105 might affect
glutamate-gated calcium channels.
We had also reported that CT 105 inhibited calcium uptake into rat
brain microsomes by
Mg2+-Ca2+ ATPase
(51)
and that CT 105 inhibits the activity of
Na+-Ca2+ exchanger in
SK-N-SH cells (52)
. Alterations in neuronal
Na+-Ca2+ exchange in AD
have been reported (53)
.
Taken together, our data suggest that increased calcium influx, inhibition of calcium efflux, and inhibition of calcium uptake into intracellular calcium storing organelles may contribute to the increase in intracellular calcium concentration and neurotoxicity induced by CT.
We also investigated the conformation of CT 105 using a CD experiment.
CT 105 has a ß-sheet conformation of ~20% in various solutions. As
listed in Table 1
, the ß-sheet content of CT 105 is larger in DPC
micelle and anionic vesicles than that in aqueous solution. Since DPC
micelle and unilamellar vesicles are good phospholipid-containing model
membranes, this implies that interactions with the cell membrane induce
more ß-sheet structure in Aß-peptide-containing CT 105, which can
induce self-aggregates similar to Aß. This conformation might have
resistance to proteolysis by protease. Monomeric
Aß1–40 or Aß1–42 is
in a random coil or -helical conformation and stimulates outgrowth
in vitro (54)
. A change into ß-sheet
conformation leads to the formation of fibrils and a concomitant toxic
effects toward neurons in vitro (54
, 55)
.
Transition to the ß-sheet conformation proceeds faster below pH 6.5
and at an increased Aß concentration (56
, 57)
.
Furthermore, fibril formation accelerates upon nucleation, when initial
fibrils have formed.
Taken together, these results might indicate that CT accumulated intra- or extracellularly by proteolytic processing of APP disrupts calcium homeostasis by different mechanisms in neuronal cells. Our unpublished data show that the CT deletion mutant without an Aß region is also toxic to differentiated PC 12 cells and rat primary cortical neurons. Thus, these data raise the possibility that the toxicity by CT 105 is caused by an additive action of carboxyl-terminal ends without Aß or Aß.
In conclusion, the demonstration for the first time of the calcium homeostasis-disrupting effect of CT 105 peptide would support the evidence for its involvement in neurodegeneration characteristic of AD. Also, it can be postulated that an altered processing or overproduction of APP could result in the intracellular production of the CT fragment, calcium homeostasis disruption, and triggering lethal process. The Aß-bearing CT fragment is not only the precursor of Aß production, but is also a more active and toxic product than Aß. Internal CT fragments may be further metabolized to Aß, which is deposited in the brain where it inflicts additional toxicity to neurons.
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ACKNOWLEDGMENTS |
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Received for publication September 2, 1999.
Revision received December 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and b) small unilamellar vesicles such as
PC vesicle (•), PC/PG (3:1) vesicle (