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,#1
* Biochemistry and
Pharmacology, School of Medicine, University of Catania, 95125 Catania;
Institute of Respiratory Disease, Ospedale Tomaselli, 95125 Catania, Italy;
§ Consorzio Mario Negri Sud, 66030 S. Maria Imbaro, Chieti, Italy;
¶ Department Internal Medicine and Medical Therapy, University of Pavia, 27100 Pavia, Italy;
Department of Pharmaceutical Sciences, School of Pharmacy, University of Catania, 95125 Catania;
# I. N. M. Neuromed, Localita‘ Camerelle, 86077 Pozzilli, Isernia, Italy
1Correspondence: Department of Pharmaceutical Sciences, Section of Pharmacology, School of Pharmacy, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. E-mail: nicoletti.ferdinando{at}ctonline.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: neuronal apoptosis • cell cycle • Alzheimer’s disease.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Degenerating neurons show the
morphological features of apoptosis (2
, 3)
. ß-Amyloid
peptide (ßAP), the main constituent of amyloid plaques
(4)
, is believed to play a causative role in the
pathogenesis of AD and induces neuronal apoptosis in vitro
(5
, 6)
. Understanding the mechanism(s) responsible for
ßAP-induced apoptosis may therefore be relevant for the
identification of novel therapeutic strategies in AD. Although numerous
mechanisms have been implicated as initial triggers of ßAP toxicity,
such as activation of surface receptors (7
8
9
10
11)
,
generation of radical oxygen species (12)
, or interference
with cell adhesion to the extracellular matrix (13
, 14)
,
the linking bridge between these initial events and the execution of
apoptosis are still obscure.
We proceeded from the observation that ßAP is toxic to neurons
(5
, 6)
, but does not affect glial cell viability
(15
16
17)
. An induction of c-jun and c-fos has been
causally linked to neuronal apoptosis induced by ßAP (18
, 19)
, and this is supported by the presence of Jun or Fos
immunoreactivity in tangle-bearing neurons of AD brain
(20)
. However, ßAP also induces c-jun and c-fos
expression in astrocytes (21
, 22)
, which are resistant to
death. Knowing that c-jun and c-fos respond to mitotic stimuli in
proliferating cells (23
, 24)
, we have hypothesized that
ßAP generates a proliferative signal, which can be afforded by
astrocytes but may not be tolerated by postmitotic cells such as
neurons.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pure neuronal culture
Cultures of pure cortical neurons were obtained from E 15 rat
embryos. Cortices were dissected in a
Ca2+/Mg2+ free buffer;
pieces were collected by slow speed centrifugation and cells were
mechanically dissociated in a plating medium consisting of DMEM/Ham’s
F12 (1:1) supplemented with the following components: 10 mg/ml bovine
serum albumin, 10 µg/ml insulin, 100 µg/ml transferrin, 100 µM
putrescine, 20 nM progesterone, 30 nM selenium, 2 mM glutamine, 6 mg/ml
glucose, 50 units/ml penicillin, and 50 µg/ml streptomycin. Cortical
cells were plated at a density of 2 x
106/dish on 35 mm Nunc dishes precoated with 0.1
mg/ml poly-D-lysine. Cytosine-ß-D-arabinofuranoside (10 µM) was
added to the cultures 18 h after plating to avoid the
proliferation of non-neuronal elements and was kept for 3 days before
medium replacement. Subsequent partial medium replacements were carried
out every 2 days. This method yields more than 99% pure neuronal
cultures, as judged by immunocytochemistry for glial fibrillary acidic
protein (GFAP) and neuron-specific microtubule-associated protein 2
(MAP-2). ßAP has always been applied to cultures at 8–12 days
in vitro.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde for 15 min at room
temperature and permeabilized with 0.1% Triton X-100 for 5 min. After
blocking for 30 min with 3% normal goat serum, incubation with
polyclonal GFAP antibodies (Sigma Immunochemicals, 1:100 dilution) was
carried out for 2 h at room temperature. After washing,
biotin-conjugated anti-rabbit secondary antibodies (7 µg/ml) were
added for 1 h. After a 45 min incubation with the ABC complex
(Vectastain ABC-Elite kit, Vector, Burlinghame, Calif.), staining was
developed by
DAB-nickel/H2O2 reaction.
Ten microscopic fields per culture were marked on the dish and
visualized on a video monitor connected to a digital video camera.
Images of the fields were examined for GFAP staining and were
computer-stored prior to MAP-2 staining. For MAP-2 staining, cultures
were blocked for 30 min with 3% horse serum and incubated for 2 h
at room temperature with monoclonal MAP-2 antibodies (Affiniti
Research, 1:1000 dilution). After washing, biotin-conjugated anti-mouse
secondary antibodies (7 µg/ml) were added for 1 h. After a 45
min incubation with the ABC complex, staining was developed by
DAB-/H2O2 reaction. Marked
fields were analyzed for MAP-2 staining and their images were compared
to those previously stored.
Handling of ßA peptides
ßAP(1–42),
ßAP(1–40),
ßAP(25–35), and the control peptide reverse
ßAP(35–25) were purchased from Bachem
Feinchemikalien AG (Bubendorf, Switzerland). Different lots of the
peptides were used. Peptides were solubilized in sterile, doubly
distilled water at an initial concentration of 2.5 mM and stored frozen
at -20°C. Before the experiment, ßAP(1–42)
and ßAP(1–40) stock solutions were kept for 1
wk in a 37°C incubator so as to allow aggregation and therefore
toxicity. ßAP(25–35) was toxic soon after
solubilization (25)
. The reverse ßAP
(35–25) was generally not toxic; however, we
observed some toxicity when it was stored frozen for long periods of
time. All peptides were used to a final concentration of 25 µM in the
presence of the glutamate receptor antagonists MK-801 (10 µM) and
DNQX (30 µM) to avoid the potentiation of endogenous glutamate
toxicity (26)
. ßAP(1–42) and
ßAP(1–40) behaved similarly to
ßAP(25–35) in activating cell cycle. For this
reason, most of the characterization has been performed with
ßAP(25–35), which more reproducibly forms
aggregates in culture.
FACS analysis
For FACS analysis cells were harvested by incubation with
0.25% trypsin for 3 min, and after addition of 50% fetal calf serum
the suspension was centrifuged at low speed. Each pellet was washed
with phosphate-buffered saline and finally fixed in 70% ethanol. Prior
to propidium iodide staining (50 µg/ml in the dark for 30 min),
suspended cells were treated for 1 h at 37°C with RNase (100
µg/ml). DNA content and ploidy were assessed by using a Coulter Elite
flow cytometer. The Multicycle AV software program (Phoenix Flow
Systems, San Diego, Calif.) was used to analyze cell cycle distribution
profiles.
For immunofluorescent staining, suspended cells, prepared as described above, were permeabilized with 0.01% triton-X100 and then stained for 1 h at 4°C with a mouse monoclonal antibody to MAP-2 (Affiniti Research Products, 1:750 dilution) or with a rabbit polyclonal phospho- retinoblastoma (RB) antibody (Medical & Biological Laboratories Co., Ltd., Tokyo, Japan; 1 µg/ml) specific for a phospho-Ser containing peptide (one-letter code: TRPPTLS(p)PIPHIP-KLH) conserved in rat, mouse, and human RB. After washing, FITC-conjugated secondary antibodies (1:50 dilution) were added for 30 min.
Thymidine incorporation
For measurement of thymidine incorporation, neuronal cultures
were labeled for 20 h with 1 µCi of
[3H]methyl-thymidine (sp. act.: 25 Ci/mmol,
Amersham, Italia). ßAP(25–35) was either
co-added or applied 8 and 16 h before ending the incubation. At
the end of the incubation, cultures were washed extensively with
ice-cold phosphate-buffered saline and incubated for 30 min in 1 N
ice-cold HClO4. After two additional washings
with 0.5 N HClO4 and a final wash with ethanol,
precipitates were solubilized with 0.5 N NaOH and aliquots were
collected for measurement of
[3H]methyl-thymidine incorporation.
Bromodeoxyuridine incorporation assay
To assess bromodeoxyuridine (BrdU) incorporation, 10 µM BrdU
(Sigma, St. Louis, Mo.) was added to neuronal cultures together with 25
µM ßAP(25–35) and cells were incubated for
18 h. Cells were then fixed for 10 min at -20°C in 95%
ethanol/5% acetic acid. DNA was denatured by incubation with 2 N HCl
for 30 min at room temperature; afterward, cultures were washed twice
(5 min each) with Sorenson’s buffer 0.4 M for neutralization of HCl.
For double-labeling experiments with anti-MAP-2 and anti-BrdU antibodies, cultures were first processed for anti-MAP-2 labeling to demonstrate that the immunostaining with anti-MAP-2 was not affected by the cell fixation and DNA denaturation procedure used for BrdU staining. For MAP-2 staining, cultures were blocked for 30 min with 3% horse serum, then incubated for 2 h at room temperature with monoclonal MAP-2 antibodies (Affiniti Research, 1:750 dilution). After washing, Cy3-conjugated anti-mouse secondary antibodies (Vector, 1:200) were incubated for 45 min. Cells were observed in an inverted fluorescence microscope with a rhodamine filter. Images of marked fields stained for MAP-2 were computer-stored before processing for anti-BrdU labeling. Cultures were blocked again for 30 min with 3% bovine serum albumin before the addition of monoclonal anti-BrdU antibodies (Sigma Immunochemicals, 1:1000 dilution) for 1 h. After washing, Cy3-conjugated anti-mouse secondary antibodies (Vector, 1:200) were incubated for 45 min and anti-BrdU immunofluorescence was revealed as described above. Marked fields were analyzed for nuclear BrdU staining and their images were compared to those previously stored. Fluorescent secondary antibodies, in the absence of BrdU primary antibodies, never labeled the nuclear region of MAP-2 stained neurons.
Immunoblotting
For Western blot analysis, neurons were harvested at 4°C in a
10 mM Tris buffer (pH 7.4) containing 5 mM EDTA, 1 mM PMSF, 25 µg/ml
leupeptin, and 0.5% aprotinin. After 10 min centrifugation at 8000
rpm, an aliquot of the supernatants was processed for the assessment of
protein concentration by a bicinchoninic acid kit. Samples were diluted
in sodium dodecyl sulfate-bromphenol blue buffer and boiled for 5 min
before loading. Electrophoresis was performed in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (30 mA/h) using 60 µg of
cell protein per lane. After separation, proteins were transferred onto
a nitrocellulose membrane (Hybond ECL, Amersham Italia) for 45 min
using a transblot semi-dry transfer cell. After blocking, membranes
were incubated with primary antibodies for 2 h at room
temperature, then repeatedly washed and exposed to horseradish
peroxidase-conjugated secondary antibodies for 1 h at room
temperature. Proteins were visualized using the enhancing
chemiluminescence detection system (ECL, Amersham Italia). The
following primary antibodies were used: mouse monoclonal cyclin D1
antibody (Santa Cruz, final dilution: 1 µg/ml); rabbit polyclonal
phospho-RB antibody (Medical & Biological Laboratories; 1 µg/ml);
rabbit polyclonal cyclin E antibody (Santa Cruz, 1 µg/ml); and rabbit
polyclonal cyclin A antibody (Santa Cruz, 1 µg/ml).
Neuronal transfection
Transfection was carried out as follows: 10 µg of plasmid
containing a cyclin D1 antisense or a dominant negative mutant of Cdk2
(both provided by A. Sala and described in Results) was equilibrated
with 20 µl Lipofectin (Life Technologies, Inc.) in 1 ml of
conditioned medium for 1 h before addition to the culture dish.
Transfection was allowed overnight before fresh medium replacement and
was carried out 36 h before ßAP(25–35)
treatment. When necessary, Dex (1 µM) was added 12 h prior to
ßAP(25–35) addition. Transfection efficiency
was quantitated by cotransfecting 10 µg of the leukocyte marker CD20
in a pCMV-neo-Bam vector. Cell surface expression of CD20 was revealed
by staining living cells with a PE-conjugated anti CD20 mouse
monoclonal antibody (Immunotech, Marseille, France; 1:10 dilution),
followed by fixation with 2% paraformaldehyde and FACS analysis.
Mock-transfected neurons were used for control of CD20 staining.
Hoechst chromatin staining in transfected cultures
Cultures were stained with the fluorescent chromatin dye,
Hoechst 33258 (0.4 µg/ml for 10 min at 37°C, after 15 min fixation
with 4% paraformaldehyde), after being transfected with either As-D1
or DN-CDK2 and exposed to ßAP(25–35) for
24 h. For the identification of neurons transfected with As-D1 (15
µg/dish), cultures were cotransfected with 5 µg/dish of
pEGFP-IRESneo vector encoding for EGFP. Neurons transfected with
DN-CDK2 were instead identified by immunostaining for an influenza
protein HA-epitope tag inserted just prior to the first stop codon of
the DN-CDK2 insert. Immunostaining was performed using an
affinity-purified rabbit polyclonal antibody raised against the
HA-epitope tag (Santa Cruz, 1 µg/ml) and a Cy3-conjugated anti-rabbit
secondary antibody (1:500, Vector Laboratories).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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of pure rat cortical astrocytes
stimulated with ßAP(25–35) showed a greater
percentage of cells in S phase (Fig. 1
), which is consistent with an activation of cell cycle.
|
To address the same issue in neurons, we first established the
conditions to grow virtually pure cultures of cortical neurons (>99%
MAP-2 positive and GFAP negative; Fig. 2
), which were healthy and viable up to 12 days in vitro
(DIV). Neuronal cultures were treated with
ßAP(1–42), ßAP(1–40),
the active fragment ßAP(25–35), or the control
reverse ßAP (35–25) always in the presence of
a mixture of ionotropic glutamate receptor antagonists. In untreated
cultures or in cultures treated with the reverse ßAP
(35–25), <1% of neurons were in S or G2/M
phase (exemplified in Fig. 3A, B
), suggesting that contamination by neuroblasts was
minimal. Addition of 25 µM ßAP(25–35) to the
cultures increased substantially the percentage of neurons in S phase,
although no cell was observed in G2/M phase of the cell cycle (Fig. 3C
). ßAP(25–35) treatment also
increased the incorporation of
[3H]-methylthymidine into DNA (Fig. 3D
). The neuronal origin of cells synthesizing DNA in
response to ßAP(25–35) is also shown by double
fluorescent staining with MAP-2 antibodies and bromodeoxyuridine (Fig. 3E, F
).
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FACS analysis revealed that in most of the cultures the percentage of
neurons found in S phase increased to ~8–10% between 8 and 16 h after addition of ßAP(25–35), and declined
to 4–5% at 20 h (Fig. 4A
). In the same cultures,
ßAP(25–35)-induced apoptosis (quantitated by
FACS as in Fig. 4C, D
) increased linearly between 8 and
20 h (Fig. 4B
). The same extent of neuronal apoptosis
was detected by Hoechst staining in sister cultures (not shown). A
similar time course for both S phase and apoptosis was observed after
incubating the cultures with ßAP(1–40) and
ßAP(1–42) (Fig. 4E, F
). No effects
were induced by ßAP(1–42) applied to the
cultures immediately after solubilization, i.e., under conditions in
which the peptide does not form toxic aggregates at least during the
first 24 h in culture (see legend of Fig. 4
). These results
therefore suggest that cortical neurons exposed to ßAP undergo
apoptosis after entering S phase. The smaller percentage of neurons in
S phase with respect to the percentage of apoptotic neurons may reflect
a rapid transfer from S phase into the apoptotic phenotype.
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Sequential expression of cell cycle proteins in ßAP-treated
neurons
To examine whether neurons exposed to ßAP enter S phase
following the typical sequence of events driving the G0-G1/S
transition, we have measured 1) the induction of cyclin D1
and phosphorylation of RB protein by activated cyclin-dependent protein
kinase 4 (Cdk4) as events associated with mid G1
phase, and 2) the induction of cyclin E and A, as markers of
late G1 and S phase, respectively (28
, 29)
. Western blot
analysis with anti-cyclin D1 antibodies revealed a 34 kDa band
corresponding to the molecular mass of cyclin D1. Expression of cyclin
D1 was low in control cultures and increased in response to
ßAP(25–35), showing a peak at 4 h. Cyclin
D1 levels were still detectable at 8 h, but markedly decreased
after 20 h of exposure to ßAP(25–35)
(Fig. 5
). The amount of phosphorylated RB (pRB) was also increased by
ßAP(25–35) with a similar time course (Fig. 5)
, suggesting an induced formation of functionally active
Cdk4/6-cyclin D1 complex leading to phosphorylation of RB and,
presumably, to the release of the transcription factor E2F
(30)
. Cyclin E, a target of E2F (31)
, and
cyclin A were also induced, but with a longer latency, increasing after
8 h of exposure to ßAP(25–35) (Fig. 5)
.
Thus, ßAP-treated neurons enter S phase through the sequential
expression of cell cycle proteins operating in proliferating cells.
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Activation of cell cycle in response to ßAP is causally related
to neuronal death
To examine the relationship between activation of cell cycle and
neuronal death, we transfected the cultures with a dexamethasone (Dex)
-inducible cyclin D1 antisense (As-D1) (32)
or a dominant
negative mutant of Cdk2 (DN-CDK2) (29)
. The As-D1 is a 606
bp murine cDNA fragment ligated in an antisense orientation with
respect to the Dex-inducible MMTV-LTR promoter contained in the
pMAM-neo vector (32)
; the DN-CDK2, in which a dominant
negative mutation was introduced by an Asp to Asn mutation at the
protein kinase KLADFGLAR consensus site, was inserted in a pCMV-neo-Bam
vector (29)
. For the control of transfection efficiency,
cultures were cotransfected with a pCMV-neo-Bam vector expressing the
leukocyte marker CD20, which was revealed by immunofluorescent
staining. Cytofluorometric analysis showed that 18–30% of neurons
were CD20 immunopositive in different experiments (exemplified in
Fig. 6
). Induction of As-D1 by 1 µM Dex reduced the percentage of neurons
entering S phase (Fig. 7A
) as well as the amount of the direct Cdk4/6-cyclin D1
target, pRB (Fig. 7C, D
), in cultures exposed to
ßAP(25–35). Control As-D1 transfection without
Dex (i.e., a condition in which As-D1 cannot be expressed) had no
effect (Fig. 7A
). CMV-mediated expression of DN-CDK2 gave
results comparable to those obtained by As-D1 + Dex (Fig. 7A
). The induced expression of both As-D1 (in the presence
of Dex) and DN-CDK2 significantly protected against
ßAP(25–35)-induced apoptosis as assessed by
cytofluorometric analysis (Fig. 7B
). In addition, neurons
expressing As-D1 after treatment with Dex always showed integrity of
nuclear chromatin as revealed by Hoechst 33258 staining in cells
coexpressing the enhanced green fluorescent protein (EGFP) (Fig. 8A, B,G
). In contrast, the majority of EGFP-positive cells from cultures
not treated with Dex (i.e., transfected but not expressing As-D1)
showed signs of chromatin fragmentation and/or condensation (Fig. 8C, D, G
). Hoechst staining also showed integrity of nuclear
chromatin in neurons expressing DN-CDK2, identified by immunostaining
for an influenza hemagglutinin (HA)-epitope tag (Fig. 8E, F, G
). Taken collectively, these results indicate that activation of
cell cycle is not only antecedent to but also causally related to
neuronal death. Because of the unexpectedly high extent of
neuroprotection in transfected cultures, we wondered whether primarily
surviving neurons might have spread the protection to other neurons in
culture through the production of soluble factors. Cultures were
therefore transfected with As-D1 ± Dex and then treated with
ßAP(25–35) for 20 h. The medium collected
from ßAP(25–35)-treated cultures expressing
As-D1 (i.e., transfected with As-D1 + Dex) was neuroprotective when
applied to receiving cultures, which in turn were treated with
ßAP(25–35). Protection was not observed with
the medium collected from cultures untreated with Dex (Table 1
). To further support a causal relationship between cell cycle induction
and neuronal death, we treated the cultures with chemical inhibitors of
the cell cycle such as mimosine (33)
, an inhibitor of
cdk’s by competing for the ATP binding domain of the kinases,
deferoxamine (34)
, a chelator of the iron required for the
onset of DNA synthesis, or transforming-growth factor ß (TGF-ß),
which induces cell cycle arrest in a number of proliferating cells
(35)
and protects human neurons against ßAP toxicity
(36)
. All these agents substantially reduced the
percentage of neurons in S phase after exposure to
ßAP(25–35) and were neuroprotective against
ßAP(25–35)-induced apoptosis (Fig. 7E, F
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Sympathetic
neurons, however, are not affected by a dominant negative form of Cdk2
(40)
, which was instead protective in our model. Thus,
activation of an unscheduled cell cycle may be common to several forms
of neuronal apoptosis, but subtle differences exist with respect to the
cell type and death paradigm. For example, as opposed to NGF-deprived
sympathetic neurons (40)
, cortical neurons challenged with
ßAP must cross the late G1/early S phase (a process that requires
Cdk2 activation) before dying. The mechanism by which ßAP-treated
neurons interrupt the cell cycle and enter the execution phase of
apoptotic death remains to be established. p53 or other potential
targets for Cdk activity may trigger apoptotic death through the
activation of effectors such as caspases, the inhibition of which is
protective against ßAP-induced toxicity (44
, 45)
.
Our finding is in line with recent papers describing the reappearance
of mitotic proteins in neurons from autoptic brain samples of AD
patients (46
47
48
49
50)
. Initially, the reported presence of
mitotic phospho-epitopes in neurons bearing neurofibrillary tangles
(46)
could be interpreted as a terminal byproduct of
apoptotic degeneration; however, more recently, cell cycle markers have
been described in susceptible regions of AD brain, but not necessarily
in late degenerating neurons (50)
. This suggests that the
activation of a cell cycle may be a requisite antecedent to neuronal
death in AD. A relationship between the mitotic cycle and the AD
phenotype has also been inferred from the evidence that presenilin 1
and 2 (which are mutated in most of early-onset familiar AD) are
present in the nuclear membrane and are associated with subcellular
structures involved in cell cycle regulation and mitosis
(51)
.
We now provide evidence that neurons begin a cycle in response to
ßAP: they complete G1, are blocked after entering S phase, and
eventually die. The lack of neurons in G2 phase in our study diverges
from the appearance of cyclin B1 or activated cyclin B1/Cdk1 complex in
AD neurons (49
, 50)
. The absence of specific environmental
factors in our cultures might have prevented progression of the cell
cycle into the G2 phase, as may occur in the AD brain. It is noteworthy
that ßAP-induced ‘neuronal cycle’ was accompanied by the sequential
expression of cell cycle proteins, because this suggests that targeted
inhibitors of these proteins may function as neuroprotectants in
AD.;1>
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Nature (London) 374,647-650[Medline]
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 M. Hamdane, A. Bretteville, A.-V. Sambo, K. Schindowski, S. Begard, A. Delacourte, P. Bertrand, and L. Buee p25/Cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death J. Cell Sci., March 15, 2005; 118(6): 1291 - 1298. [Abstract] [Full Text] [PDF]
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