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The Journal of Neuroscience, December 1, 1998, 18(23):9790-9799
Overexpression in Neurons of Human Presenilin-1 or a Presenilin-1
Familial Alzheimer Disease Mutant Does Not Enhance Apoptosis
Sherry
Bursztajn1,
Richard
DeSouza2,
Donna L.
McPhie3,
S. A.
Berman1,
Junichi
Shioi4,
Nikolaos K.
Robakis4, and
Rachael L.
Neve3
1 Department of Psychiatry and Program in Neuroscience,
Harvard Medical School, Belmont, Massachusetts 02478, 2 Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, 3 Department of
Genetics, Harvard Medical School, McLean Hospital, Belmont,
Massachusetts 02478, and 4 Department of Psychiatry and
Fishberg Research Center for Neurobiology, Mount Sinai School of
Medicine, New York, New York 10029
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ABSTRACT |
Programmed cell death, or apoptosis, has been implicated in
Alzheimer's disease (AD). DNA damage was assessed in primary cortical neurons infected with herpes simplex virus (HSV) vectors expressing the
familial Alzheimer's disease (FAD) gene presenilin-1 (PS-1) or an FAD
mutant of this gene, A246E. After infection, immunoreactivity for PS-1
was shown to be enhanced in infected cells. The infected cells
exhibited no cytotoxicity, as evaluated by trypan blue exclusion and
mitochondrial function assays. Quantitative analysis of cells that were
immunohistochemically labeled using a Klenow DNA fragmentation assay or
the TUNEL method revealed no enhancement of apoptosis in PS-1-infected
cells. This result was confirmed using assays for chromatin
condensation and for DNA fragmentation. Expression of PS-1 protected
against induction of apoptosis in the cortical neurons by etoposide or
staurosporine. The specificity of this phenotype was demonstrated by
the fact that cortical cultures infected with recombinant HSV vectors
expressing the amyloid precursor protein (APP-695) showed, in contrast,
a significant increase in the number of apoptotic cells and an increase
in DNA fragmentation for all parameters tested. Our results indicate
that overexpression of wild-type or A246E mutant PS-1 does not enhance
apoptosis in postmitotic cortical cells and suggest that the previously
reported enhancement of apoptosis by presenilins may be dependent on
cell type.
Key words:
apoptosis; neurodegeneration; presenilin; amyloid
precursor protein; DNA fragmentation; Alzheimer's disease
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INTRODUCTION |
Cell death can occur by two
morphologically and biochemically distinct pathways: necrosis and
apoptosis. These two pathways are not mutually exclusive, and both
types of death have been observed in the brain in Alzheimer's disease.
Necrosis is characterized by cell swelling, rapid lysis of the cellular
membrane, and expulsion of intracellular organelles. In contrast, in
apoptosis, or programmed cell death, nuclear changes and alterations in
chromatin material are produced by a series of well orchestrated
cellular events that require RNA and protein synthesis. Apoptotic cells
show morphological changes that include condensation and fragmentation
of heterochromatin, membrane blebbing, loss of the nuclear envelope,
and cellular fragmentation into apoptotic bodies, whereas most of the
organelles remain intact. During apoptosis, a specific endonuclease
cleaves the DNA between nucleosomes into characteristic fragments that form a ladder pattern when separated on an agarose gel, whereas in
necrosis, DNA becomes degraded randomly.
Familial Alzheimer's disease (FAD) can be caused by mutations in the
amyloid precursor protein (APP) gene or in the presenilin genes PS-1
and PS-2. Overexpression in neuronal cells of FAD APP cDNAs that are
mutated at V642 (the "London" mutation) has been shown to cause
double-stranded DNA breaks (Yamatsuji et al., 1996 a,b), one
feature of the programmed cell death termed apoptosis (Bredesen, 1995;
Thompson, 1995). Recently, two genes that cause early onset FAD, S182
[presenilin-1 (PS-1)] and STM2 [presenilin-2 (PS-2)], have been
identified (Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington
et al., 1995). Approximately half of inherited AD cases are
caused by mutations in these two genes. It has been reported that
overexpression of these genes in transfected cell lines can cause
apoptosis (Janicki and Monteiro, 1997) or result in an increased
susceptibility to apoptosis (Deng et al., 1996; Guo et al., 1996, 1997,
1998; Wolozin et al., 1996). However, it is not known whether the
overexpression of wild-type or FAD mutant PS-1 in postmitotic cortical
neurons results in DNA damage or apoptosis. To gain a better
understanding of the role of these genes in neuronal apoptosis, we have
used herpes simplex virus (HSV) vectors to overexpress wild-type and
mutant PS-1 cDNAs in primary cortical neurons. We show here that
apoptosis is not enhanced in neuronal cells infected with these
vectors, and we suggest that the previously reported enhancement of
apoptosis by presenilins may be cell type-dependent.
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MATERIALS AND METHODS |
Generation of recombinant HSV vectors and infection of
primary mouse cortical cultures. The human PS-1 cDNA was obtained
by PCR screening (Chow et al., 1996) of a human fetal brain cDNA library. The QuikChange mutagenesis kit (Stratagene, La Jolla, CA) was
used to generate the A246E mutation in the PS-1 cDNA. The presence of
the mutation was verified by DNA sequence analysis.
We prepared replication-defective HSV vectors expressing the wild-type
(HSV/PS-1) and mutant (HSV/A246E-PS-1) human PS-1 cDNAs and
Escherichia coli  -galactosidase (HSV/Lac; negative
control) in the expression vector pHSVPrpUC as described (Carlezon et
al., 1997). Some experiments used an HSV vector expressing N-terminal myc epitope (EQKLISEEDL)-tagged PS-1 cDNA. HSV/APP-695 was prepared as
described (McPhie et al., 1997). The titer of the helper virus component of each stock was 1-1.2 × 106
plaque-forming units (pfu)/ml on 2-2 cells. The titer of the recombinant virus component of each stock, as assayed by expression of
the exogenous gene in PC12 cells, was consistently 3 × 107 infectious units (iu)/ml.
Cortices from embryonic day 18 (E18) mice (Charles River, Wilmington,
MA) were dissociated mechanically in serum-free Neurobasal medium
supplemented with B27 (Life Technologies, Gaithersburg, MD). Cells were
plated at a density of 3 × 106 cells/ml in
poly-L-ornithine-coated 35 mm culture dishes and were
maintained in Neurobasal medium supplemented with B27 and 1% each
fetal bovine and horse serum. Cells were infected with HSV recombinants
5 d after plating. Immunoblots (see Fig. 2) to demonstrate levels
of expression of the HSV recombinant genes used primary cultures from
E18 rat embryos that were generated and maintained under the same
conditions as the mouse primary neuronal cultures.
Determination of efficiency of expression of HSV
recombinants. Efficiency of infection of the control virus HSV/Lac
was determined using X-gal histochemistry to detect cells expressing
-galactosidase (LacZ). Twenty-four hours after infection, the
percentage of LacZ-positive cells was determined by comparing the
number of blue cells with the total number of cells in 10 random
microscope fields for each multiplicity of infection (moi).
Immunocytochemistry with a monoclonal antibody to the myc tag (9E10;
American Type Culture Collection) was used to determine the efficiency
of infection of neurons by HSV/myc-PS-1. Twenty-four hours after
infection, the cells were fixed with 4% paraformaldehyde at room
temperature for 20 min, washed with PBS, and incubated with a 1:50
dilution of the primary antibody. The myc epitope was localized with a
secondary antibody conjugated to horseradish peroxidase (HRP), which
was visualized by incubating the cells in the 3,3'-diaminobenzidine
(DAB) substrate for 5 min. Cells on coverslips were rinsed with
PBS, mounted onto glass microscope slides using 90% glycerol in PBS,
and viewed with bright-field optics.
Immunoblot analysis of APP-695 and PS-1 expression in infected neurons
was performed as described (Elder et al., 1996). For APP-695 detection,
20 µg of protein was loaded into each lane of a 7% Tris-glycine gel.
The mouse monoclonal anti-APP antibody 22C11 (Boehringer Mannheim,
Indianapolis, IN) was used at a 1:1000 dilution. For PS-1 detection, 40 µg of protein was loaded into each lane of a 10-20% Tris-tricine
gel. The rabbit polyclonal 347 antiserum, against PS-1 amino acids
2-12, was used at a 1:10,000 dilution. The blots were processed using
the SuperSignal immunodetection system (Pierce, Rockford, IL).
Cell viability assays were performed to determine whether the HSV
viruses affected cell survival. Cell viability was measured both by
metabolic conversion of tetrazolium salt into formazan salt using a
Cell Titer 96 Assay Kit (Promega, Madison, WI) and by using a trypan
blue exclusion test.
Detection of DNA fragmentation using a photometric enzyme
immunoassay. The presence of cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) was quantitatively determined after induced cell death via Cell Death Detection ELISA (Boehringer Mannheim). Cells were ruptured in lysis buffer, vortexed, and incubated for 30 min. The lysate was centrifuged at 200 × g for 10 min, after which the supernatant was transferred
into wells of a streptavidin-coated microtiter plate. An
immunocomplex consisting of anti-histone-biotin (a biotinylated mouse
monoclonal antibody, which bridges the histone component of the
nucleosomes and the streptavidin coat on the plates) and anti-DNA-POD
(a peroxidase-conjugated mouse monoclonal antibody, which reacts with
the DNA component of the nucleosomes) was added to the plate, which was
incubated at room temperature for 2 hr. The plates were washed to
remove unbound antibodies, and the amount of histone-associated DNA
fragments was quantified by analysis of the POD retained in the
immunocomplex using a 2,2'-azino-di(3-ethylbenzthiazolin-sulfonate)
substrate, which was detected photometrically at a wavelength of 405 nm.
Detection of DNA strand breaks using a Klenow fragment
end-labeling technique. An alternative method of detecting DNA
fragmentation involves histochemical labeling of exposed 3'-OH ends
of the DNA using the FragEL-Klenow DNA Fragmentation Detection kit
(Oncogene Research Products). Cultured cells on coverslips were fixed
as described previously (Didier et al., 1996), washed, incubated in TBS
for 15 min at room temperature, permeabilized with proteinase K, and
treated with H2O2 to inactivate endogenous
peroxidases. The cells were incubated in a humidified chamber at 37°C
for 1.5 hr with biotinylated deoxynucleotides and Klenow enzyme,
washed, and incubated with streptavidin-HRP, which was then visualized using DAB as substrate. Coverslips were mounted onto glass slides in
90% glycerol/PBS and were viewed with bright-field microscopy.
Detection of DNA double-strand breaks using the TUNEL in
situ apoptosis assay. The TUNEL method (Gavrieli et al.,
1992), which detects dioxigenin-labeled genomic DNA in situ
using a modified terminal transferase dUTP nick end labeling (TUNEL)
technique, was used to detect DNA double-strand breaks. The procedure
was performed as described previously (Didier et al., 1996), using an
Apoptag in situ apoptosis detection kit (Oncor).
Detection of apoptotic nuclear morphology using
bisbenzimide. Bisbenzimide binds to and allows visualization of
chromatin. Bisbenzimide (1 µg/ml) was added to the medium in which
the fixed cells on coverslips were mounted onto glass slides, and cells were viewed with fluorescent microscopy to visualize the nuclear morphology, as described previously (Bruner and Bursztajn, 1986; Didier
et al., 1996). The number of apoptotic nuclei, which appear smaller
than normal and in which the chromatin appears condensed, were
counted and compared with the total number of cells in 10 random fields
for each moi.
Isolation of nuclear and cytoplasmic DNA from cultures infected
with HSV recombinants. Nuclear and cytoplasmic DNA was purified from three 35 mm dishes per sample of infected neurons, 24 hr after
infection, as described by Greenberg and Ziff (1984). After electrophoresis of the DNAs on agarose gels, they were transferred to
Biotrans membrane (ICN Biochemicals, Costa Mesa, CA) and probed with
radiolabeled total mouse DNA to increase the sensitivity of detection
of DNA fragmentation.
Induction of apoptosis. Twelve hours after infection with
HSV vectors, cells were exposed to 20 µM etoposide or 1 µM staurosporine for 5 hr. DNA fragmentation was detected
using the photometric enzyme immunoassay described above.
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RESULTS |
Determination of optimal efficiency of infection with
recombinant HSV
HSV/Lac, expressing the E. coli -galactosidase gene,
was used to determine the optimal conditions for gene transfer into the
mouse cortical neurons. E17-18 neurons were infected with HSV/Lac at
5 d in vitro (DIV) using a range of moi from 0.1-5.0 as described (McPhie et al., 1997). Twelve hours after infection, X-gal
histochemistry was performed to visualize cells expressing the
-galactosidase transgene. Infection with HSV/Lac at an moi of 2 (Fig. 1A) yielded an
efficiency of expression that was very near that of cultures infected
with HSV/Lac at an moi of 5 (Fig. 1B). Quantitation
of -galactosidase-positive cells shows that at an moi of 0.1 almost
no cells expressing LacZ were detected, whereas at 1, 2, and 5 moi, 85, 90, and 95%, respectively, of cells were lacZ positive (Fig.
1C).
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Figure 1.
High levels of efficiency of expression are seen
after recombinant HSV infection of cortical neurons. Mouse cortical
neurons were maintained in Neurobasal medium with B27 supplement and
1% horse and fetal bovine serum. At 5 DIV, cells were infected with
recombinant HSV viruses at a range of moi. A-C, HSV/Lac
infection of primary cortical neurons at an moi of 2 (A) or 5 (B). Twelve hours
after infection, cells were fixed and processed for -galactosidase
activity (blue cells). C, LacZ-positive
and total cells were counted in 10 random fields for each moi, and the
percentage of blue cells was determined. The data are expressed as the
average ± SE of 10 microscopic fields, with a total of 553-641
cells counted for each moi. D-G, HSV/myc-PS-1 infection
of primary cortical neurons at an moi of 1 (D) or
5 (E). The brown reaction product delineates myc
immunoreactivity in infected cells. F, G,
Uninfected neurons incubated with Myc antibody and processed in
parallel with the cells shown in D and E.
D-F are bright-field images. G is a
phase-contrast image of F. Scale bar, 25 µm.
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A similar efficiency of expression was seen when the cultures were
infected with HSV/myc-PS-1, expressing PS-1 with an N-terminal myc tag
(Fig. 1D-G). Cells were infected with HSV expressing
myc-PS-1, and the myc tag was immunodetected with the 9E10 monoclonal
antibody, using DAB as the chromogen. The majority of cells in cultures infected with HSV/myc-PS-1 (Fig. 1D,E) displayed myc
immunoreactivity. The myc immunoreactivity was specific, in that
uninfected neurons incubated with the 9E10 antibody and processed
simultaneously with cells shown in Figure 1D,E show
virtually no immunoreactivity (Fig. 1F,G). Immunoblot
analysis (Fig. 2B)
demonstrated overexpression of full length PS-1 or A246E-PS-1 in
primary neuronal cultures infected with an moi of 0.5 or 1 of the
appropriate virus. Infection of parallel cultures with an HSV vector
expressing the amyloid precursor protein APP-695 (Fig.
2A) demonstrated similar overexpression of
APP-695.
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Figure 2.
PS-1 and APP-695 overexpression in cortical
cells. Primary neuronal cells prepared from E18 rat cortex were
infected with recombinant HSV expressing APP-695, wild-type PS-1, or
A246E-mutant PS-1, or were mock-infected. A, Cell
extracts from cortical neurons that were infected with HSV/APP-695
(lanes 1 and 2, moi = 0.5 and 1, respectively) or HSV/Lac (lanes 3 and 4,
moi = 0.5 and 1, respectively) or that were mock-infected (lanes 5 and 6) were analyzed by immunoblot analysis using 22C11 (Boehringer
Mannheim). The cluster of bands around 110 kDa represent immature and
mature (differentially glycosylated) APP-695. B, Cell
extracts from cortical neurons that were infected with HSV/PS-1
(lanes 1 and 2, moi = 0.5 and 1, respectively), HSV/A246E-PS-1 (lanes 3 and
4, moi = 0.5 and 1, respectively), or HSV/Lac
(lanes 5 and 6, moi = 0.5 and 1, respectively) or that were mock-infected (lanes 7 and
8) were analyzed by immunoblot analysis using the rabbit
polyclonal 347 antiserum, against PS-1 amino acids 2-12. Full-length
PS-1 and the N-terminal fragment (NTF) are
immunodetected at 49 and 29 kDa, respectively. The band at 34 kDa
probably is nonspecific. Detection of the full-length PS-1 bands was
inhibited by the cognate peptide and was heat-sensitive (data not
shown). At the exposure times used, endogenous full-length PS-1 was not
detected.
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Cellular viability after infection with
HSV recombinants
To assess potential cytotoxicity in response to infection of
neurons with HSV recombinants expressing -galactosidase, PS-1, or
A246-PS-1, cortical neurons infected at a range of moi were assayed for
trypan blue exclusion and mitochondrial function. Cortical neurons
infected with HSV/PS-1 or HSV/A246E-PS-1 and incubated with 0.2%
trypan blue 24 hr after infection showed no staining with the dye,
which is taken up only by cells whose membrane is compromised (Fig.
3A-F). Trypan blue
exclusion does not rule out apoptosis, because loss of membrane
integrity occurs very late during apoptosis, but it does indicate that
the cells are viable.
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Figure 3.
Cortical neurons infected with HSV/PS-1 (A,
D), HSV/A246E-PS-1 (B, E), or HSV/Lac (C,
F) at an moi of 2 are viable. Twenty-four hours after
infection, neurons were tested for exclusion of trypan blue. All
HSV-infected neurons showed exclusion of trypan blue.
A-C are phase-contrast images. D-F are
bright-field images.
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Further confirmation of the health of the cells is seen in the
microscopic images (Fig. 3A-C), which show phase bright
cells with long neuronal processes. In an independent assay for the viability of the HSV-infected cells, we quantified cellular survival after HSV/PS-1 infection, using the
3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyl tetrazolium bromide
metabolic assay. Neurons were infected in parallel with vehicle (mock)
or with HSV vectors expressing wild-type or A246E-mutated PS-1, or
LacZ, at moi of 0.1-5 (Fig.
4A-C). No significant
difference between groups in cellular survival was observed up to 72 hr. It can be concluded, therefore, that overexpression of wild-type or
mutated PS-1 via HSV vectors is not sufficient to induce cell death in
cortical neurons.
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Figure 4.
HSV-mediated expression of wild-type or mutated
presenilins does not affect neuronal survival. Neurons were infected in
parallel with HSV expressing wild-type PS-1, A246 PS-1, or LacZ, or
vehicle only (Mock) at moi ranging from 0.1 to 5. Survival was measured by metabolic conversion of tetrazolium salt into
formazan salt using a Cell Titer 96 Assay Kit. A, Twelve
hours after infection; B, 24 hr after infection;
C, 72 hr after infection. A representative result from
one of three independent experiments is shown. Each value is the
average of three different wells, with error bars representing the
range.
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Characterization of DNA damage in neurons infected with
HSV recombinants
We previously reported a form of DNA damage that is characterized
by single-stranded DNA breaks, with no detectable double-stranded DNA
breaks (Didier et al., 1996). Frequent single- and double-stranded DNA
breaks are detected in DNA during apoptosis (Peitsch et al., 1993).
Neurons with single-stranded DNA breaks are viable and may represent an
earlier stage of apoptosis than neurons with double-stranded DNA
fragmentation. We performed four different assays to determine whether
PS-1 overexpression induces single- or double-stranded DNA breaks as
seen in apoptosis.
The Klenow fragment of DNA polymerase is used in an assay that
preferentially detects single-stranded DNA breaks (Rosl, 1992; Lauc et al., 1994). The polymerase binds to the exposed 3'-OH ends of
DNA fragments that are generated when DNA strands are nicked, and it
catalyzes the template-dependent addition of a mixture of
biotin-labeled and unlabeled deoxynucleotides (Lauc et al., 1994).
Cortical cultures that were infected for 24 hr at an moi of 2 with HSV
recombinants expressing wild-type or A246-mutated PS-1, or lacZ, or
that were mock-infected, show few cells with detectable single-stranded
DNA breaks, which are indicated by darkly stained nuclei (Fig.
5A,B,D,E). In sharp contrast,
cells infected with HSV/APP695 show an increase in cells with apoptotic nuclei (Fig. 5C), despite the fact that their neuronal
processes remain intact. These latter data are consistent with the
recent report that overexpression of APP-695 in hippocampal neurons
in vivo causes nuclear DNA fragmentation (Nishimura et al.,
1998). Quantitative analyses of the data (Fig.
6A,B) confirm that
overexpression of wild-type or A246E mutant PS-1 does not enhance
apoptosis in cortical neurons, whereas apoptosis is increased
significantly (p < 0.05; Bonferroni multiple
t test) in neurons infected with HSV/APP-695.
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Figure 5.
Single-stranded DNA breaks in cells of recombinant
HSV-infected cultures were assessed using a Klenow assay. The dense
reaction product (arrows) identifies apoptotic nuclei.
Control cells incubated without the Klenow enzyme give negative results
(no staining). Cortical cultures infected for 24 hr at an moi of 2 with
HSV/PS-1 (A) or HSV/A246E-PS-1
(B) show very few apoptotic nuclei. Cultures
infected under the same conditions with HSV/APP-695
(C) show numerous apoptotic nuclei.
D, LacZ-infected cells; E, mock-infected
cells.
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Figure 6.
Quantification of the data shown in Figure 5 shows
that overexpression of wild-type or A246E-mutant PS-1 does not enhance
apoptosis in cortical cells, as measured with a Klenow DNA
fragmentation assay. However, cells infected with HSV/APP-695 show a
significant increase (p < 0.05; Bonferroni
multiple t test) in percentage of apoptotic cells.
Cortical cells were infected as described in the legend to Figure 5.
The total number of cells and the number of apoptotic cells were
counted in 10 random fields, and the number of apoptotic cells was
expressed as a percentage of the total cells counted. The results are
the average ± SEM. A total of 200-300 cells were counted for
each moi. * denotes a significant (p < 0.05) difference in the percentages of apoptotic cells in
HSV/APP-695-infected cultures versus HSV/Lac-infected cultures.
A, Twelve hours after infection; B, 24 hr after
infection.
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This lack of DNA fragmentation in PS-1-infected cells, and the enhanced
apoptosis in cortical cells infected with HSV/APP695, was also seen
when we used a DNA fragmentation enzyme immunoassay that detects
histone-associated DNA fragments, thereby revealing internucleosomal
degradation of genomic DNA that occurs during apoptosis (Compton, 1992;
Pandey et al., 1994; Stewart, 1994; Allen et al., 1997). Neurons
infected with HSV/APP-695, but not neurons infected with the other HSV
recombinant vectors, showed significant increases in DNA fragmentation
(p < 0.05) (Fig.
7A,B).
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Figure 7.
Quantification of apoptosis using a DNA
fragmentation enzyme-immunoassay (Cell Death Detection ELISA), which
detects histone-associated DNA fragments that are generated during the
internucleosomal degradation of genomic DNA that occurs in apoptosis.
Neurons were infected in parallel with HSV/PS-1, HSV/A246E-PS-1,
HSV/APP-695, or HSV/Lac, or vehicle only (mock) at moi ranging from 1 to 5. Samples were analyzed photometrically at 405 nm, using substrate
solution as a reference blank. Only neurons infected with HSV/APP-695
show significant changes in DNA fragmentation. A, Twelve
hours after infection; B, 24 hr after infection. Results
are the average of three different wells; error bars represent
range.
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TUNEL staining was also used to visualize apoptosis
immunocytochemically (Gavrieli et al., 1992). This method, in contrast to the Klenow fragmentation assay, detects primarily double-stranded DNA breaks via terminal deoxynucleotidyl transferase (TdT) addition of
deoxynucleotides to the 3'-OH ends of the DNA strands (Didenko and
Hornsby, 1996; Didier et al., 1996). Cortical cells that were infected with HSV vectors expressing wild-type or A246E-mutated PS-1,
or lacZ, or that were mock-infected show only a few TUNEL-positive cells (Fig. 8A,B,D,E),
whereas cells infected with HSV/APP695 show numerous apoptotic nuclei
(Fig. 8C). Quantification of the data revealed that a
significant increase over control in the number of apoptotic cells was
observed only in HSV/APP-695-infected cells (p < 0.05). Cultures infected with all other HSV recombinants showed no
significant difference from control mock-infected cells in number of
TUNEL-positive nuclei (Fig.
9A,B).
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Figure 8.
Overexpression of wild-type or A246E-mutated PS-1
does not enhance apoptosis in cortical cells, as detected with a TUNEL
method (Apoptag). This method, in contrast to the Klenow fragmentation
assay, detects primarily double-stranded DNA breaks. Cortical cells
were infected as described in the legend to Figure 5. Cells were fixed
and incubated with anti-digoxigenin antibodies coupled to peroxidase,
and the brown reaction product was detected with DAB. For each
experiment, nonspecific labeling was determined by omitting TdT. Few
apoptotic cells were detected (arrows) 24 hr after
infection with HSV expressing wild-type (A) or
mutated (B) PS-1. An increased in apoptotic cells
was observed in cells infected with HSV expressing APP-695
(C). Cells infected with HSV/Lac
(D) or incubated with the vehicle
(E, mock) show very few apoptotic cells.
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Figure 9.
Quantification of the percentages of apoptotic
cells shown in Figure 8. Apoptotic cells were counted as described in
the legend to Figure 7. Enhanced apoptosis was not observed after
infection of the neurons with HSV/PS-1 or HSV/A246E-PS-1, but it was
observed when the cells were infected with HSV/APP-695.
A, Twelve hours after infection; B, 24 hr
after infection. The results are the average ± SEM of counts from
10 microscopic fields with a total of 200-300 cells counted per moi. *
denotes significance (p < 0.05) in the
difference in values between cells infected with HSV/APP-695 and cells
infected with HSV/Lac.
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We further studied the nuclear morphology of cortical cells after
infection with HSV recombinants by staining infected cells with
bisbenzimide. Bisbenzimide binds to chromatin, allowing fluorescent visualization of normal and condensed chromatin (Bruner and Bursztajn, 1986). Morphologically, cells undergoing apoptosis show chromatin condensation, loss of nuclear envelope, and cellular fragmentation into
apoptotic bodies (Cohen et al., 1992; Didier et al., 1996). The
bisbenzimide assay yielded results similar to those obtained with the
Klenow polymerase, DNA fragmentation, and TUNEL assays. Cortical cells
that were infected with HSV vectors expressing wild-type PS-1, mutated
(A246E) PS-1, or lacZ, or that were mock-infected show only few nuclei
with altered morphology (Fig.
10A,B,D,E), whereas
cells infected with HSV/APP-695 show an increase in the number of
nuclei having condensed chromatin material (Fig. 10C). Quantitation of apoptotic cells detected with bisbenzimide showed that
only cells infected with HSV/APP-695 have a significant increase in the
number of apoptotic cells 24 hr after infection (Fig.
11A,B) (p < 0.05 by Bonferroni t test).
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Figure 10.
Bisbenzimide intercalates into DNA strands,
allowing for fluorescent visualization of normal or condensed
chromatin. Normal nuclei are regular in shape and round, whereas
apoptotic nuclei (arrows) show condensation of chromatin
material. Cortical cells infected with HSV/PS-1
(A) or HSV/A246E-PS-1 (B)
for 24 hr at an moi of 2 show infrequent alterations in nuclear
morphology. Cells infected with HSV/APP-695 (C)
show an increase in the number of nuclei with condensed chromatin
material. Cells infected with HSV/Lac (D) or
incubated with vehicle alone (E) show similar
nuclear morphology as those infected with HSV/PS-1 or
HSV/A246E-PS-1.
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Figure 11.
Quantification of apoptotic cells with
bisbenzimide shows a lack of enhanced apoptosis 24 hr after infection
with HSV/PS-1 or HSV/A246E-PS-1. Enhanced apoptosis was observed 24 hr
after infection with HSV/APP-695 at an moi of 2. The total number of
cells and the number of nuclei with condensed chromatin were counted in
10 random fields, and each value was expressed as the percentage of
nuclei with condensed chromatin relative to the number of total nuclei
counted. The results are the average ± SEM. A total of 400-500
cells were counted for each moi. * denotes significance
(p < 0.05) in the difference in values
between cells infected with HSV/APP-695 and cells infected with
HSV/Lac. A, Twelve hours after infection;
B, 24 hr after infection.
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Nucleosomal and cytoplasmic DNA isolated from the infected cells did
not display an apoptotic ladder pattern when separated by agarose gel
electrophoresis, even when a Southern blot using total mouse DNA as
probe was performed to increase sensitivity (data not shown). The lack
of detectable DNA laddering in the HSV/APP-695-infected cells suggests
that this may not be as sensitive an assay for DNA fragmentation as the
in situ assays that were used to detect apoptosis.
Characterization of DNA damage in neurons infected with HSV
recombinants and induced to undergo apoptosis
To determine whether presenilins predispose to apoptosis, we
tested the effect of overexpression of PS-1 or A246E-mutated PS-1 on
neurons treated with the apoptosis-inducing agents etoposide and
staurosporine (Fig. 12). Cortical cells
were infected with HSV/PS-1, HSV/A246E-PS-1, or HSV/Lac at an moi of 2. Twelve hours later, the infected cells were exposed to 20 µM etoposide or 1 µM staurosporine for 5 hr, after which the cells were evaluated with the DNA fragmentation
enzyme immunoassay that detects histone-associated DNA fragments (Fig.
7e). Both etoposide and staurosporine induced a significant increase in
DNA fragmentation in cortical cells that were mock-infected or infected
with HSV/Lac. This increase in DNA fragmentation was prevented if the
cells were infected with HSV/PS-1 or with HSV/A246E-PS-1 before being
exposed to the apoptosis-inducing agents.
View larger version (29K):
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Figure 12.
Expression of PS-1 or A246E-PS-1 in cortical
neurons protects against apoptosis induced with etoposide or
staurosporine. Twelve hours after infection of cortical cells with
HSV/PS-1, HSV/A246E-PS-1, or HSV/Lac at an moi of 2, cells were exposed
to etoposide (ET, 20 µM) or staurosporine
(Stau, 1 µM) for 5 hr. In an additional
control, mock-infected cells were exposed to the same drugs. Samples
were analyzed photometrically as described in Figure 7. Both etoposide
and staurosporine induced a modest, albeit significant increase in DNA
fragmentation in cortical cells (p < 0.05).
This increase in DNA fragmentation was prevented if cells were infected
with HSV/PS-1 or with HSV/A246E-PS-1 before being exposed to the
apoptosis-inducing agents.
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DISCUSSION |
Multiple laboratories have reported evidence for apoptosis, or
programmed cell death, in pathological tissues obtained from the brains
of Alzheimer's disease patients (Su et al., 1994; Guo et al., 1998).
Therefore it is of interest that overexpression of FAD mutants of APP
(Yamatsuji et al., 1996a,b; Giambarella et al., 1997; Zhao et al.,
1997) or of wild-type APP itself [Nishimura et al. (1998); this
report] causes increased apoptosis, and that overexpression of PS-1
and PS-2 in transfected cell lines results in an increased
susceptibility to apoptosis (Deng et al., 1996; Guo et al., 1996;
Wolozin et al., 1996), possibly by perturbing cellular calcium
regulation and promoting oxidative stress (Guo et al., 1997, 1998).
The question of whether these genes induce apoptosis in primary
neurons, however, has remained unanswered. PS-1 function may differ in
primary neurons from cell lines. PS-1 is reported to be expressed
primarily in CNS neurons in the brain, suggesting that this protein may
perform a neuron-specific function (Elder et al., 1996). In fact, in
AD, neurons that express PS-1 antigen are less vulnerable to the
disease than are neurons that do not express it (Giannakopoulos et al.,
1997), and inhibition of PS-1 expression results in apoptosis (Roperch
et al., 1998), suggesting a protective role for this protein. We
therefore sought to determine whether overexpression of wild-type or
A246E-mutated PS-1 induces DNA damage or apoptosis in primary mouse
neurons in culture.
We used HSV vectors expressing wild-type or A246E-mutant PS-1, as well
as wild-type APP-695 and appropriate controls, to determine whether
overexpression of these proteins leads to enhanced apoptosis in primary
cortical neurons. We showed that cortical neurons infected with HSV
vectors are viable and that when we infect at an moi of 2, the majority
of the cortical neurons express the appropriate HSV-encoded protein. We
also demonstrated that the overexpression of wild-type or A246E mutant
PS-1 does not result in enhanced apoptosis. This was shown with four
independent assays: (1) the Klenow DNA fragmentation assay for
single-stranded DNA breaks; (2) a test to detect histone-associated DNA
fragments; (3) the TUNEL technique; and (4) bisbenzimide detection of
chromatin condensation. Finally, we showed that overexpression of PS-1
or PS-1-A246E in cortical neurons suppresses apoptosis induced by
etoposide or staurosporine.
In contrast to the results obtained with PS-1, overexpression of
APP-695 in cortical neurons enhanced apoptosis. We tested APP-695 for
its effect on apoptosis in neurons, because this spliced form of APP is
expressed preferentially in neurons (Neve et al., 1988). In PC12
cells transfected with wild-type APP, TUNEL-positive cells were
increased almost twofold relative to controls (Zhao et al., 1997). We
show that overexpression of APP in primary neurons gives an even
greater enhancement of apoptosis over controls, a result consistent
with the recent demonstration by Nishimura et al. (1998) that
overexpression of wild-type APP-695 in rat neurons in vivo
causes nuclear DNA fragmentation in transfected cells. Notably, the
magnitude of APP-695-induced apoptosis was lower at 24 hr than at 12 hr
after infection. Although we do not know the reason for this, it would
be interesting to determine whether this decrease is caused by activity
of cellular DNA repair mechanisms.
In contrast to results that have implicated presenilins in enhancing
apoptosis or in rendering various cell lines more susceptible to
apoptosis (Deng et al., 1996; Wolozin et al., 1996; Guo et al., 1997;
Janicki and Monteiro, 1997), our results with cortical neurons
show that overexpression of wild-type or A246E-mutated PS-1 is not
sufficient by itself to induce apoptosis. In fact, overexpression of
these proteins protects cortical neurons from experimentally induced
apoptosis. However, at least 30 different missense mutations and one
in-frame splice site mutation in PS-1 have been reported to cause FAD
(Cruts et al., 1996), and we do not know whether all of these mutants
are unable to induce apoptosis in primary neurons or whether they have
a protective effect against apoptosis. In any case, the ability
of PS-1 to induce apoptosis appears to be cell-type specific, and this
may have important implications for the pathogenesis in AD, in which
neurons are differentially affected.
| |
FOOTNOTES |
Received April 23, 1998; revised Sept. 11, 1998; accepted Sept. 11, 1998.
This work was partly supported by the Massachusetts Alzheimer's
Disease Research Center (S.B.), by the Alzheimer Disease and Related
Disorders Association (N.K.B.), and by National Institute on Aging
Grants AG12954 (R.L.N.) and AG08200 (N.K.R.). We thank Dr. Robert
Coopersmith for invaluable help with the figures.
Correspondence should be addressed to Dr. Sherry Bursztajn at her
present address: Biomedical Research Institute F6-21, Louisiana State
University Medical Center, 1501 Kings Highway, Shreveport, LA 71130, E-mail: sbursz{at}lsumc.edu, or Dr. Rachael L. Neve at her present
address: MRC, McLean Hospital, 115 Mill Street, Belmont, MA 02478. E-mail: neve{at}helix.mgh.harvard.edu
Dr. Berman's present address: Louisiana State University Medical
Center, Shreveport, LA 71130.
| |
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Abstract
Introduction
