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The Journal of Neuroscience, May 1, 2000, 20(9):3104-3114
Involvement of Retinoblastoma Family Members and E2F/DP Complexes
in the Death of Neurons Evoked by DNA Damage
David S.
Park1, 4,
Erick
J.
Morris2,
Rod
Bremner3,
Elizabeth
Keramaris1,
Jaya
Padmanabhan4,
Michele
Rosenbaum4,
Michael L.
Shelanski4,
Herbert M.
Geller2, and
Lloyd A.
Greene4
1 Neuroscience Research Institute, University of
Ottawa, Ottawa, Ontario, K1H 8M5, Canada,
2 Department of Pharmacology, University of Medicine and
Dentistry of New Jersey-Robert Wood Johnson Medical School,
Piscataway, New Jersey 08854, 3 Eye Research Institute of
Canada, University of Toronto, Toronto, Ontario, M5T 2S8, Canada, and
4 Department of Pathology and Center for Neurobiology and
Behavior, Columbia University College of Physicians and Surgeons, New
York, New York 10032
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ABSTRACT |
Neuronal death evoked by DNA damage requires cyclin-dependent
kinase 4 (Cdk4) and 6 activity and is accompanied by elevation of cyclin D1-associated kinase activity. Because Cdk4/6
phosphorylates retinoblastoma protein (pRb) family members that
then modulate the transcriptional activity of E2F/DP1 complexes,
we examined the involvement of these components in DNA damage-evoked
neuronal death. Camptothecin induced rapid pRb and p107 phosphorylation at a Cdk4/6 phosphorylation site followed by selective loss of Rb and
p107. The CDK inhibitor flavopiridol suppressed pRb and p107
phosphorylation and loss, implicating CDK activity in these events.
Moreover, the loss of pRb and p107 appeared to be mediated by caspases
because it was blocked by general caspase inhibitors. The role of
phosphorylation and pRb and p107 loss in the death pathway was
indicated by observations that virally mediated expression of pRb
mutated at sites of phosphorylation, including the Cdk4/6 site,
inhibited death. Finally, expression of dominant-negative versions of
DP1, known to compromise E2F transcriptional activity, protects
cortical neurons from death induced by camptothecin and sympathetic
neurons from death evoked by UV treatment. Taken together, these
results implicate the CDK-pRb/E2F/DP pathway as a required element in
the neuronal death evoked by DNA damage.
Key words:
apoptosis; pRb; E2F; DP1; neuronal; cyclin
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INTRODUCTION |
Although neuronal apoptosis is an
important component of brain development (Oppenheim, 1991 ), a growing
body of evidence suggests that it also participates in the progression
of neuropathological conditions such as stroke and neurodegenerative
disease (Anderson et al., 1996; Stefanis et al., 1997; Chen et al.,
1998; Cotman, 1998). Although numerous etiologies and putative
death initiators have been described, the signaling pathways that
control neuronal death are not fully understood.
Multiple lines of evidence indicate that cell cycle elements play an
important role in some but not all paradigms of neuronal death. For
example, NGF deprivation leads to increased cdc2 activity and cyclin B
expression in neuronal pheochromocytoma 12 (PC12) cells (Gao and
Zelenka, 1995), as well as elevated cyclin D1 transcript levels in
sympathetic neurons (Freeman et al., 1994). Others observe increases in
cell cycle molecules in affected regions of brains from Alzheimer's
disease patients and in brains exposed to ischemic conditions (McShea
et al., 1997; Nagy et al., 1997; Vincent et al., 1997; Busser et al.,
1998). In addition, cyclin D1-associated kinase activity is elevated in
cortical neurons treated with the DNA-damaging agent camptothecin (Park
et al., 1998b). With respect to functional inhibition studies, we have
shown that G1/S blockers (Farinelli and Greene, 1996) and the
CDK inhibitors suppress the death of sympathetic and cortical neurons
evoked by trophic factor deprivation (Park et al., 1996a) and/or
DNA-damaging conditions (Park et al., 1997a, 1998a). Finally, in the
latter systems, virally mediated expression of cyclin-dependent kinase
(CDK) inhibitors such as p16 and p27 or of dominant-negative forms of
Cdk4 and 6 but not Cdk2 or 3 inhibit death (Park et al., 1997b,
1998b).
Our observations on the importance of Cdk4/6 activity in certain
paradigms of neuronal death raise the questions of the target(s) of
this activity and whether such a target(s) plays required roles in the
death mechanism. The only presently known in vivo substrates of activated Cdk4/6 are the tumor suppresser pRb and its related family
member p107 (Chellappan et al., 1991; Beijersbergen et al.,
1995; Weinberg, 1995). Although pRb appears to interact with numerous
proteins, its regulation of the cell cycle-related transcription factor
E2F is best understood. pRb is normally hypophosphorylated in quiescent
cells such as neurons and binds to and inhibits the transcriptional
activity of E2F. In cells undergoing G1-to-S cell cycle transition, pRb
becomes hyperphosphorylated. Numerous studies indicate that a
consequence of Rb hyperphosphorylation is the release and activation of
E2F (Chellappan et al., 1991; Suzuki-Takahashi et al., 1995).
The E2F family of transcription factors has a diverse set of cellular
functions (Nevins, 1992; La Thangue, 1994) that include the control of
cell proliferation. In this case, transcriptional activation requires
association of E2F with its obligate binding partner DP1-2 (Bandara et
al., 1993; Girling et al., 1993; Wu et al., 1996).
In addition to cell cycle progression, there are also reports
implicating Rb and E2F in apoptosis. For example, Rb null mice show
neurological deficits and neuronal apoptosis (Jacks et al., 1992;
Macleod et al., 1996). Furthermore, overexpression of E2F in
proliferating cells results in apoptosis (Qin et al., 1994; Hiebert et
al., 1995). Finally, there is evidence that E2F, DP1, and their
complexes interact with and regulate other proteins such as MDM2
and p53 (Martin et al., 1995; O'Connor et al., 1995; Sorensen et al.,
1996).
These observations lead us to the hypothesis that the neuronal death
evoked by DNA damage is mediated via Cdk4/6 activation, consequent pRb
phosphorylation, loss of pRb function, and activation of E2F/DP
complexes. To test this model, we determined whether changes in pRb
phosphorylation occur during the death of camptothecin-treated neurons.
In addition, we examined the potential changes in the expression levels
of cell cycle control elements including CDKs, cyclins, and Rb family
members. Finally, we examined the potential role of E2F/DP in the death
of neurons evoked by DNA damage.
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MATERIALS AND METHODS |
Materials.
( )cis-5,7-Dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)piperidinyl]-4H-benzopyran-4-one
(L86-8275; flavopiridol) was a generous gift from Dr. Peter J. Worland
(National Cancer Institute). Camptothecin was obtained from Sigma (St.
Louis, MO). Boc-aspartyl- (ome)-fluoromethyl ketone (BAF) and
zVAD-fluoromethylketone (zVAD-fmk) were purchased from Enzyme
Systems Products (Dublin, CA). Adenovirus expressing  -galactosidase
(LacZ) was a kind gift from Dr. Ruth Slack (University of Ottawa).
Generation of recombinant Sindbis viruses. The cDNAs for the
coding regions of wild-type (WT) human DP1, dominant-negative (DN) ( 103-126) DP1 (containing a deletion of the
DNA-binding domain), the DN (1-126) DP1 truncation mutant
containing a deletion of amino acids 1-126, and (233-272) DP1
containing a deletion of the E2F-binding domain (233-272) were
inserted into the BSTE11 site of the DSTEQ12 vector (Joe et al., 1996;
Park et al., 1997c) The dominant-negative forms of DP1 have been
reported previously to inactivate the transcriptional activity of E2F
(Wu et al., 1996). These would be expected to interfere with complexes
of all E2F/DP family members. FLAG tags
(ATGGACTACAAGGACGATG-ATGACAAA) were introduced at the 3' end of the
coding region of WT DP1, DN (103-126) DP1, and (233-272) DP1.
Control nonexpressing vectors of each DP1 construct were generated by
eliminating the initiating codon. All mutations, deletions, and FLAG
tags were introduced by PCR and confirmed by sequencing. Viral
particles were generated by in vitro transcription and
transfection into BHK cells and titered by plaque assay as
described previously (Joe et al., 1996).
Generation of recombinant adenovirus. The cDNA for the
coding regions of the K11 Rb mutant (full-length mouse Rb containing T246 to A, T350 to R, S601 to A, S605 to A, S773 to A, S781 to A, S788
to A, S800 to A, S804 to E, T814 to A, and T819 to A mutations; a kind
gift from Dr. Eldad Zacksenhaus, University of Toronto) (Brown et al.,
1999) or green fluorescent protein (GFP) was subcloned into pAdlox
vector (Hardy et al., 1997) and recombined with adenoviral backbone DNA
in CRE8 cells as described previously (Hardy et al., 1997). The
K11 Rb construct was derived from P34Rb (Hamel et al., 1992) by
the addition of three additional phosphorylation site mutations (S773,
T814, and T819 to A mutations).
Culture and survival of cortical neurons. Mouse (embryonic
day 16 for Rb expression experiments) or rat (embryonic day 18) cortical neurons were cultured as described previously (Park et al.,
1998b) and grown in defined serum-free medium. These cultures typically
contain >99% neurons as assessed by staining with antibody directed
against phosphorylated neurofilament (RT97; Developmental Studies
Hybridoma Bank, University of Iowa). The neurons were plated into
6-well dishes (~3,000,000 cells/well) or 24-well plates (200,000 cells/well) coated with poly-L-lysine (100 µg/ml) in serum-free medium [N2:EMEM (1:1) supplemented with 6 mg/ml
D-glucose, 100 µg/ml transferrin, 25 µg/ml insulin, 20 nM progesterone, 60 µM putrescine, and 30 nM selenium]. For Westerns (6-well dishes), the neurons
were treated with camptothecin (10 µM) alone or with flavopiridol (1 µM), BAF (100 µM), or
zVAD-fmk (100 µM) in serum-free medium 2-3 d after
plating. At appropriate times of culture under the conditions described
in the text, cells were washed twice in PBS and collected by
trituration in PBS. The neurons were centrifuged in an Eppendorf
tabletop microfuge for 3 min at 10,000 rpm. The neurons were then lysed
in sample buffer as described previously (Park et al., 1998b).
Alternatively, neurons were infected 1 d after plating (in 24-well
plates) with Sindbis virus at a multiplicity of infection (MOI) of ~2
or with adenovirus at an MOI of 150 and incubated overnight. The medium
was then exchanged with serum-free medium supplemented with 10 µM camptothecin where appropriate. At the indicated times
of culture under the conditions described in the text, cells were
lysed, and the numbers of viable cells were evaluated as described
previously (Park et al., 1998b). Briefly, cells were lysed in 200 µl
of cell lysis buffer [0.1× PBS, pH 7.4 (0.4 mM
Na2HP04, 0.15 mM KH2P04, 13.5 mM NaCl, and 0.25 mM KCl)] containing 0.5%
Triton X-100, 2 mM MgCl2, and
cetyldimethylethylammonium bromide (0.5 gm/100 ml) that disrupts cells
but leaves the nuclei intact. Ten microliters of sample from each
culture were loaded onto a hemacytometer, and the number of healthy
intact nuclei was evaluated by phase microscopy. Nuclei that displayed
characteristics of blebbing, disruption of nuclear membrane,
phase-bright apoptotic bodies, and chromatin margination were excluded.
All experimental points are expressed as a percentage of the cells
plated on day 0 and are reported as the mean ± SEM
(n = 3).
Rat sympathetic neuron culture and survival assay. Primary
cultures of sympathetic neurons were generated from dissociated superior cervical ganglia of postnatal day 1 Sprague Dawley rats as
described previously (Park et al., 1996b). The cells were plated in 0.5 ml of medium per well in collagen-coated 24-well dishes at a density of
~0.5 ganglia per well (~10,000 neurons/well). The growth medium was
RPMI 1640 medium supplemented with 10% heat-inactivated horse serum
and 60 ng/ml mouse NGF (Sigma). To eliminate non-neuronal cells, we
added a mixture of uridine and 5-fluorodeoxyuridine (10 µM each) to the cultures on the following day. On the
third day after plating, the neurons were infected with Sindbis virus (1-2 plaque-forming units per cell) in 0.2 ml of RPMI 1640 medium containing 2% heat-inactivated horse serum. After 1 hr of infection, 0.3 ml of RPMI 1640 medium containing 16% heat-inactivated horse serum
was added. The cultures were then left to incubate overnight before UV
irradiation (300 J/m2). Neurons were UV
irradiated in 200 µl of medium containing NGF using a Stratolinker
(Stratagene, La Jolla, CA). After irradiation, an additional 300 µl
of medium containing NGF was added to each well. At the indicated
times, the numbers of viable, phase-bright neurons were determined by
strip counting as described previously (Park et al., 1996b). Briefly, a
24-well plate was placed onto an adjustable stage. From a defined point
(consistent throughout the course of the experiment) the same field of
neurons (consisting of the diameter of each well) was assessed by phase
microscopy. Phase-bright, intact neurons were scored as viable, whereas
those with apoptotic bodies or phase-dark shrunken cell bodies were counted as dying or dead. All experimental points were performed in
triplicate and are reported as the mean ± SEM. All points are expressed relative to the number of neurons originally plated.
Western blot analyses. The neurons or rat fibroblasts were
harvested in sample buffer as described above. Twenty-five micrograms of protein were loaded onto 10% SDS-polyacrylamide gels (except for
the p16 analysis in which a 15% gel was used) and transferred onto
nitrocellulose membrane as described previously (Park et al., 1998b).
Blots were probed with primary antibody [cyclin D1, cyclin A, cyclin
E, p107, Cdk2, Cdk3, Ckd4, Cdk6, p16, p21, and p27 from Santa Cruz
Biotechnology, Santa Cruz, CA (1:300); phospho-Rb (ser795) from New
England Biolabs, Beverly, MA (1:1000); pRb from PharMingen, San Diego,
CA (1:1000); anti--actin (1:5000) from Sigma, and anti-FLAG (1:40)
from VWR Scientific)] in PBS supplemented with 3% BSA overnight.
Blots were washed four times for 25 min each in PBS containing 0.2%
Tween 20. The membranes were then probed with the appropriate secondary
antibody (Amersham, Arlington Heights, IL, or Bio-Rad, Hercules, CA)
for 1 hr in PBS containing 5% milk. The membranes were visualized by
ECL detection (Amersham).
Immunofluorescence. Sympathetic neurons or cortical neurons
were dissociated and cultured, as described above, in six-well plates
at a density of 2 ganglia/well (sympathetic neurons) or 1,000,000 cells/well (cortical neurons). After various times of infection with
the indicated viruses, neurons were fixed with 100% ethanol for 20 min
at 20°C or 4% paraformaldehyde for 30 min at 4°C and
incubated with anti-FLAG primary antibody (1:500 dilution) or anti-pRb
antibody (1:500 dilution). FITC-conjugated horse anti-mouse (1:50
dilution) or indocarbocyanine-conjugated goat anti-mouse (1:200
dilution) was used as the secondary antibody.
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RESULTS |
pRb/p107 is phosphorylated and rapidly lost during the death of
cortical neurons evoked by camptothecin
We demonstrated previously that camptothecin (10 µM)
induces apoptotic death of cortical neurons maintained in serum-free defined medium (Morris and Geller, 1996; Park et al., 1998a,b). In this
death paradigm, cyclin D1-associated kinase activity rises greatly
within 1 hr after camptothecin treatment (Park et al., 1998b). This
increase is before caspase 3-like activation that is detectable ~4 hr
after camptothecin treatment and morphological death that is observed
10-12 hr after drug treatment (Morris and Geller, 1996; Stefanis et
al., 1999). Because of the increase in cyclin D1-associated kinase
activity and the inhibition of camptothecin-evoked neuronal death by
pharmacological CDK inhibitors or by expression of CDK
inhibitors (CKIs) or DN Cdk4/6 (Park et al., 1997a, 1998b), we
have suggested that Cdk4/6 activity may be required for death signaling
in this paradigm. Accordingly, we examined whether Rb phosphorylation
increases during a time course of camptothecin treatment by Western
blot analyses using an antibody directed against the phosphoepitope of
pRb and p107 equivalent to S795 of human pRb, a site known to be
phosphorylated by Cdk4/6. The antibody does not, however, distinguish
between the phosphorylation of pRb or p107, and so we refer to the
detected species as pRb/p107. As a control, the antibody detects the
increase in pRb/107 phosphorylation in fibroblasts after they are
switched from low- to high-serum conditions (data not shown). As shown in Figure 1, camptothecin treatment
elicits a rapid phosphorylation of pRb/p107. This phosphorylation peaks
in intensity at ~1-2 hr after the start of camptothecin treatment.
The time course of pRb/p107 phosphorylation closely follows the time
course of cyclin D1-associated kinase activity that also is greatly
elevated at 1 hr after the start of camptothecin addition (Park et al.,
1998b).
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Figure 1.
pRb/p107 is phosphorylated during the death of
cortical neurons evoked by camptothecin. A, Top, Western
immunoblots (probed with anti-phospho-Rb antibody) of whole-cell
lysates of cortical neurons after various periods of treatment with
camptothecin (10 µM). Bottom, Cotreatment
with flavopiridol (1 µM) is indicated.
campto, Camptothecin. B, Densitometric
analyses of Western immunoblots of cortical neurons treated with
camptothecin alone or cotreated with flavopiridol and probed with
anti-phospho-Rb antibody. All data points are the mean ± SEM of
data from three separate experiments and are expressed relative to the
initial amount of phosphorylated pRb/p107 at time 0.
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The CDK inhibitor flavopiridol inhibits Cdk4 activity and protects
cortical neurons from camptothecin-induced death (Park et al., 1997a).
As such, we predicted that cotreatment of neurons with flavopiridol and
camptothecin should block the phosphorylation observed with
camptothecin treatment alone. As shown in Figure 1, flavopiridol
cotreatment inhibited the camptothecin-induced induction of pRb/p107 phosphorylation.
We next examined the levels of pRb and p107 during camptothecin
treatment of cortical neurons. As shown in Figure
2, the levels of pRb and p107 are
significantly reduced after 3-4 hr of camptothecin treatment. The
observed losses of pRb and p107 are not caused by generalized loss of
proteins because little or no reduction of other cell cycle proteins
(see below) or -actin (Figs. 1, 2) is observed under the same
conditions. Moreover, this loss is inhibited by cotreatment with
flavopiridol, thus indicating that inhibition of CDK activity blocks
the degradation of pRb and p107. Such observations raise the
possibility that pRb/p107 phosphorylation in this system might target
these proteins for degradation.
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Figure 2.
pRb and p107 levels decrease during the death of
cortical neurons evoked by camptothecin. A, Top, C, Top,
Representative Western immunoblots of whole-cell extracts of cortical
neurons treated with camptothecin for the indicated times and probed
with antibodies for pRb (A) and p107
(C). Middle,
Bottom, Cotreatment with flavopiridol (1 µM; middle) and BAF (100 µM;
bottom) is indicated. Blots were stripped and reprobed
for -actin as a loading control. B, D, Densitometric
analyses of Western immunoblots of cortical neurons treated with
camptothecin alone or cotreated with flavopiridol or BAF and probed for
pRb (B) or p107 (D). All
data points are the mean ± SEM of data from three separate
experiments and are expressed relative to the initial amount of protein
at time 0.
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We observed recently that caspase activity is detectable in cortical
cultures ~4 hr after the start of camptothecin treatment (Stefanis et
al., 1999). This is similar to the time at which losses of pRb and p107
are observed. In addition, general caspase inhibitors significantly
delay death in this experimental paradigm (Stefanis et al., 1999). To
evaluate the possible involvement of caspase activity in the observed
loss of pRb and p107, we cotreated cultures with camptothecin and
caspase inhibitors. BAF (Fig. 2) and zVAD-fmk (data not shown)
significantly inhibited the loss of pRb and p107. Exposure to
flavopiridol, BAF, or zVAD-fmk alone had no effect on the levels of pRb
or p107. These results indicate that caspase activation contributes
either indirectly or directly to the degradation of pRb and p107.
Early changes in the cyclins, CDKs, and CKIs are not observed in
camptothecin-treated cortical neurons
The inhibition of camptothecin-induced neuronal death by CDK
inhibitors, along with the observed increase in cyclin D1-associated kinase activity and pRb/p107 phosphorylation, suggests the importance of Cdk4/6 signaling in the control of neuronal apoptosis. We therefore examined the expression of additional cell cycle regulators during camptothecin-evoked cortical neuronal death. As shown in Figure 3, the levels of cyclin D1 and cyclin E
did not change during camptothecin treatment. Cyclin A was undetectable
(data not shown). No difference in expression levels was detected with
flavopiridol, zVAD-fmk, or BAF cotreatment or with flavopiridol, zVAD,
or BAF alone. These results indicate that elevation of cyclin D1 levels could not account for the increase in observed cyclin D1-associated kinase activity.
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Figure 3.
Levels of cyclins, CDKs, and CKIs do not increase
during the death of cortical neurons evoked by camptothecin.
A C, Western immunoblot analyses of whole-cell extracts
of cortical neurons treated with camptothecin for the indicated times
and probed with the following antibodies: cyclin D1 and cyclin E
(A); Cdk2, Cdk4, and Cdk6
(B); and p16, p21, and p27
(C). For Cdk2 blots, an equal
amount (25 µg) of rat fibroblast cell extract was used as a positive
control.
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We next explored the expression levels of CDKs in camptothecin-treated
cortical neuron cultures. Detection of Cdk2 was inconsistent, but when
it was observed by Western blot analyses as shown in Figure 3, the
levels were significantly less than that in proliferating rat
fibroblast cultures. The levels of Cdk2 protein did not rise in
cultures treated with camptothecin and, when detectable, decreased ~4
hr after treatment. Similarly, levels of Cdk4 and 6, which were readily
detectable, did not change significantly during camptothecin-induced cortical neuronal death. As with cyclins, no significant change in the
protein levels of Cdk4 and 6 was observed in cultures cotreated with
flavopiridol, zVAD-fmk, or BAF or treated with flavopiridol, zVAD, or
BAF alone (data not shown).
Finally, changes in the levels of the CKIs p16, p21, and p27 were not
observed during the first 4 hr of camptothecin treatment and, if
present, only occurred considerably later (Fig. 3). Taken together, our
results indicate that loss of pRb and p107 is not generalized to other
cell cycle regulators and that activation of cyclin D1-associated
kinase activity and subsequent pRb/p107 phosphorylation cannot be
accounted for by changes in the levels of the cell cycle regulators examined.
Expression of pRb suppresses the death of cortical neurons evoked
by camptothecin
Our observations raise the possibility that phosphorylation and
loss of pRb/p107 play important roles in the apoptotic death of
cortical neurons evoked by camptothecin. If this is the case, we would
predict that exogenous expression of pRb would inhibit such death. To
test this, we used recombinant adenovirus to express a mutant of pRb
(K11 Rb) in which 11 of the 16 putative phosphorylation sites are
mutated (Brown et al., 1999). Among the sites mutated is the putative
Cdk4/6 phosphorylation site (Kitagawa et al., 1996). Expression in the
neurons was confirmed by Western blot analyses and immunofluorescence
(Fig. 4). Approximately 61% of the
neurons in cultures infected with K11 Rb-expressing adenovirus showed increased Rb immunoreactivity (n = 243). By
Western blot analyses, we observed expression at four to five times the
levels of endogenous pRb, and this expression was maintained for at
least 10 hr after camptothecin treatment (Fig. 4). As shown in Figure 5, expression of K11 Rb significantly
blocked death at 16 hr of camptothecin exposure [~52 vs 18%
survival in the noninfected or GFP-infected or LacZ-infected (data not
shown) controls]. Protection was transient. Minimal protection
(18 ± 2 vs 3 ± 1% in the controls) was observed at 30 hr,
and no protection was observed 48 hr after camptothecin treatment (data
not shown). Thus, these results along with our observations of
increased pRb/p107 phosphorylation and loss of pRb/p107 suggest that
pRb members may play an important regulatory role in the death of
cortical neurons evoked by DNA damage.
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Figure 4.
Immunofluorescence (A-F)
and Western immunoblot (G) analyses of K11 Rb
expression. AF, Immunofluorescence (A, C,
E) staining with an antibody directed against pRb or
corresponding light micrographs (B, D, F) of
cortical neurons in culture infected with K11 Rb-expressing virus
(C-F) or control (A, B). Neurons
were fixed and stained with anti-pRb antibody 24 hr after infection
(A-D) and after 12 hr of camptothecin treatment
(E, F). G, Western immunoblot
analysis of whole-cell extracts of cortical neurons infected with
adenovirus expressing K11 Rb (no treatment or 10 hr camptothecin
treatment) or LacZ as indicated. The blots were analyzed
using an anti-pRb antibody, stripped, and reprobed with
anti--actin.
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Figure 5.
Expression of K11 Rb suppresses the death of
cortical neurons evoked by camptothecin. Neuronal cultures were
uninfected or infected for 24 hr with adenovirus expressing K11 Rb
or GFP before camptothecin treatment for 16 hr. Each point is the
mean ± SEM of combined data from three independent experiments
(each independent experiment performed in triplicate) and is expressed
relative to the number of neurons present at the initial time of
camptothecin treatment. * indicates significance
(p < 0.02, Student's t
test) compared with GFP-infected cultures treated with
camptothecin.
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Expression of DN DP1 inhibits the death of neurons evoked by
DNA damage
Because pRb family members bind to and affect the transcriptional
activity of E2F/DP complexes, we next explored whether such complexes
play a role in the death of cortical neurons evoked by DNA damage. This
was accomplished by expression of dominant-negative versions of the
E2F-binding protein DP1 using the Sindbis virus. We have shown
previously that recombinant Sindbis virus can be used for heterologous
gene expression and the study of neuronal death mediated by NGF
deprivation and DNA damage (Park et al., 1997b, 1998b). DP1 is required
for E2F activation (Bandara et al., 1993; Girling et al., 1993; Wu et
al., 1996), and Wu et al. (1996) have shown that expression of mutant
forms of DP1 that lack the DNA-binding domain either by truncation of
126 amino acids at the N terminal (1-126 DN DP1) or by internal
deletion of the DNA-binding domain (103-126 DN DP1) mediates
inhibition of E2F-mediated transactivation and cell cycle progression.
It was also reported that a DP1 mutant with a deletion of the
E2F-binding domain (233-272 DP1) has no such dominant-negative
effect (Wu et al., 1996), and this construct was therefore used as one
negative control in our experiments.
We first tested whether expression of either (1-126) DN DP1 or
(103-126) DN DP1, both of which contain a DNA-binding domain deletion, inhibits the death of cortical neurons evoked by DNA damage.
Parallel cultures were infected with control viruses. All constructs
except the truncation DN mutant (1-126) DN DP1 were FLAG epitope
tagged (F) to permit detection of the expressed protein. Expression was
confirmed by immunofluorescence and Western blot analyses (Fig.
6) in which comparable expression levels
were observed for DN and control DP1 constructs. Approximately 61% of
neurons were FLAG immunopositive for (103-126) DN DP1
(n = 178), 63.4% of neurons were positive for
233-272 DP1 expression, and 80% were positive for WT DP1
(n = 147). Expression of all three DP1 constructs was
both cytoplasmic and nuclear. As shown in Figure
7, camptothecin alone produced 90% death
at 24 hr of treatment. Expression of each of the DN versions of DP1
significantly inhibited camptothecin-induced death (50-70% survival
with DN DP1 expression). Infection with control viruses expressing
(233-272) DP1F, WT DP1, or "stop" sequences that contain
deletions of the initiation codon to prevent protein expression had
small, but consistent, protective effects in comparison with cultures
treated with camptothecin alone, but the level of protection was
significantly lower than that achieved with the DN constructs. Cortical
neurons protected by DN DP1 expression show the flat phase-dark
morphology typical of healthy cells, whereas cultures treated with
camptothecin alone or with camptothecin and a control virus show
membrane blebbing and numerous ghost-like bodies (Fig.
8). Neuritic processes, however, did not
appear to be spared in camptothecin-treated cultures protected by DN
DP1 expression (Fig. 8).
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Figure 6.
AF, Immunofluorescence (A,
C, E) or corresponding phase (B, D, F)
images of neurons infected with control virus (A, B) or
with virus encoding (103-126) DN DP1F (C-F).
Neurons were fixed and stained with anti-FLAG antibody 24 hr after
infection (A-D) and after 12 hr of camptothecin
treatment (E, F). G, Western
immunoblot analyses of whole-cell extracts of cortical neurons infected
with Sindbis viruses expressing WT or the indicated mutant
FLAG-tagged DP1 constructs. control indicates samples in
which the neurons were infected with a control virus. The
blots were analyzed using an anti-FLAG antibody. A
nonspecific immunoreactive band is marked as indicated.
Neurons were lysed 24 hr after infection with the indicated
viruses.
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Figure 7.
Expression of DN DP1 suppresses the death
of cultured cortical neurons treated with camptothecin. Control viruses
for each vector were generated by removal of the start codon from the
insert. F denotes that the protein has a FLAG epitope
attached to the C terminal. Effects of expression of (1-126) DN
DP1, a dominant-negative mutant containing an N-terminal truncation
from amino acids 1-126 (A),
(103-126) DN DP1F, a FLAG-tagged dominant-negative mutant
containing a DNA-binding domain deletion of amino acids 103-126
(B), (233-272) DP1F, a FLAG-tagged
mutant containing the E2F-binding domain deletion of amino acids
233-272 (C), and WT DP1 and respective controls
(D) on the survival of cortical neurons after
camptothecin treatment for 1 d. A, Graph in which
each point is the mean ± SEM of combined data from three
independent experiments (each independent experiment performed in
triplicate) and is expressed relative to the number of neurons present
in each culture at the initial time of camptothecin (10 µM) treatment. BD, Representative
experiments in which each point is the mean ± SEM of three
cultures from the same experimental trial. Similar results
(B-D) were obtained in two independent
experiments.
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Figure 8.
Phase-contrast micrographs of cortical neurons
treated for 1 d with the following: no additives
(A), camptothecin (10 µM;
B), (1-126) DN DP1 virus + camptothecin
(C), and (1-126) DN DP1 control virus + camptothecin (D).
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We next examined whether death in a another model of DNA damage could
also be blocked by expression of DN mutants of DP1. As shown in Figure
9, expression of either (1-126) DN
DP1 or (103-126) DN DP1 inhibits the death of sympathetic neurons
evoked by UV irradiation (~85% survival with DN DP1 expression
vs 50% without). Expression of a mutant of DP1 [(233-272) DP1]
bearing the E2F-binding domain deletion or of WT DP1 or infection with stop control viruses that contain deletions of the initiation codon to prevent expression of DP1 proteins had no significant effects
on the kinetics of neuronal death (Fig. 9; data not shown). Sympathetic
neurons protected by DN DP1 expression showed the typical phase-bright
morphology and relatively intact neuritic processes of viable neurons.
In contrast, sympathetic neurons treated with UV irradiation alone or
after infection with control viruses displayed shrunken, phase-dark
somas and degenerating processes (Fig.
10). Expression of the
FLAG-tagged DP1 constructs was confirmed by immunofluorescence analysis
(Fig. 11).
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Figure 9.
Expression of DN DP1 suppresses the death of
cultured sympathetic neurons evoked by UV irradiation. Control viruses
for each vector were generated by removal of the start codon from the
insert. F denotes that the protein has a FLAG epitope
attached to the C terminal. Effects of expression of (1-126) DN
DP1, a dominant-negative mutant containing an N-terminal truncation
from amino acids 1-126 (A), and of
(103-126) DN DP1F, a FLAG-tagged dominant-negative mutant
containing a DNA-binding domain deletion of amino acids 103-126,
(233-272) DP1F, a FLAG-tagged mutant containing the E2F-binding
domain deletion of amino acids (233-272), and wild type (WT) DP1
(B) on the survival of sympathetic neurons after
UV irradiation. A, Graph in which each point is the
mean ± SEM of combined data from three independent experiments
(each independent experiment performed in triplicate) and is expressed
relative to the number of neurons present in each culture at the
initial time of UV treatment. cont, Control.
B, Representative experiment in which each point is the
mean ± SEM of three cultures from the same experimental trial.
Similar results were obtained in two independent experiments.
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Figure 10.
Phase-contrast micrographs of rat
sympathetic neurons maintained in NGF-containing medium and exposed to
and/or infected with the following: no treatment
(A), UV (B), (1-126) DN
DP1 virus + UV (C), (1-126) DN DP1 control
virus + UV (D), or (1-126) DN DP1 virus alone
(E). The photos were taken 2 d after
irradiation and/or infection.
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Figure 11.
Immunofluorescence (A-C)
staining with an antibody directed against the FLAG epitope or
corresponding light micrographs (D-F) of
sympathetic neurons in culture infected with Sindbis virus expressing
(233-272) DP1F (A, D) or (103-126) DN DP1F
(B, E) or containing a nonexpressing control virus
(C, F). Neurons were stained 2 d after
infection.
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DISCUSSION |
The potential role of cell cycle elements in neuronal apoptosis is
perhaps paradoxical considering the terminally differentiated state of
neurons. However, growing evidence has suggested that elements that
normally control cell cycle progression may modulate death in a
cellular environment not conducive to proliferation. Our previous
evidence indicated a role for Cdk4/6 in the mechanism of neuronal death
evoked by DNA damage and trophic factor deprivation (Park et al.,
1997b, 1998b). Accordingly, we examined (1) the involvement of
pRb/p107, substrates for Cdk4/6, in neuronal death evoked by DNA damage
and (2) whether E2F/DP complexes play a required role in the death
signal downstream of Cdk4/6.
Phosphorylation and loss of pRb/p107
We have shown recently that camptothecin induces rapid (within 10 min) DNA strand breaks in cortical neurons (Morris et al., 1997)
and is a convenient model for examining the mechanisms that control DNA
damage-induced neuronal death. We examined here the status of key cell
cycle regulatory proteins during apoptosis of cortical neurons evoked
by camptothecin and demonstrate that pRb/p107 is phosphorylated during
such treatment. pRb phosphorylation has also been observed in cultures
of cis-platinum-treated sensory neurons (Gill and Windebank,
1998) and in cerebellar granule neurons deprived of
K+ (Padmanabhan et al., 1999). We also
observed here that phosphorylation of pRb/p107 is blocked by the CDK
inhibitor flavopiridol that is known to block Cdk4/6 activity
(Filgueira de Azevedo et al., 1996). Moreover, this phosphorylation
occurs early (within 1 hr) after the start of camptothecin treatment
and correlates well with the induction of cyclin D1-associated kinase
activity demonstrated previously (Park et al., 1998b). In addition, we
find that levels of pRb and p107 are greatly diminished 3-4 hr after
camptothecin treatment. This loss is inhibited by flavopiridol,
suggesting a relationship between pRb/p107 phosphorylation and loss. In
addition, zVAD and BAF also block the loss, raising the possibility
that caspases may mediate pRb and p107 degradation. pRb is a substrate of caspases and contains a consensus caspase cleavage site at its C
terminal (Tan et al., 1997). However, it is unclear whether the
observed loss of pRb/p107 is directly or indirectly mediated by caspases.
To test whether pRb and p107 are important for maintaining
survival, we forced expression of a variant of Rb that contains mutations in several phosphorylation sites including the Cdk4/6 consensus site and found that this delays the death of neurons evoked
by camptothecin. In other studies, expression of a caspase cleavage-resistant mutant of pRb abrogated TNF- -induced apoptosis of
Rb/ fibroblasts (Tan et al., 1997). The importance of Rb in cell
death and survival is underscored by observations that its
overexpression in various contexts is protective against apoptosis (Berry et al., 1996; Fan et al., 1996), whereas loss of Rb or of Rb
activity facilitates death of both non-neuronal (Liu and Kitsis, 1996;
Shan et al., 1996) and neuronal cells (Jacks et al., 1992; Lee et al.,
1992; Macleod et al., 1996). Taken together, these results suggest that
the maintenance of the levels and/or activity of Rb members plays an
important role in neuronal death evoked by DNA damage.
Levels of cyclins, CDKs, and CKIs
As discussed above, we found previously that Cdk4/6 increases
within 1 hr of camptothecin treatment. How does this activation occur?
In certain systems, it has been suggested that this regulation occurs
via modulation of levels of cell cycle regulators such as the cyclins
and CDKs. However, regulation of CDK activity can occur via multiple
mechanisms (Pines, 1993). These include (1) binding to an obligate
activating cyclin partner, (2) inhibition by endogenous CKIs, and (3)
phosphorylation at sites that are either activating or inhibitory.
Accordingly, the observed increase in cyclin D1-associated kinase
activity and the consequent pRb/p107 phosphorylation could have
occurred as a result of increased cyclin or CDK levels or by a decrease
in levels of CKIs. However, no such modulation of cyclin, CDK, or CKI
levels was detected during the early period of camptothecin treatment.
In only a few cases, for example, with p21, did the levels diminish
significantly, and these were too late to account for the early
induction of cyclin D1-associated kinase activity. Accordingly, these
observations, along with the rapid induction of cyclin D1-associated
activity (Park et al., 1998b) and pRb/p107 phosphorylation, suggest
that activation of Cdk4/6 during the death of cortical neurons evoked by DNA damage could be caused by post-translational modifications such
as phosphorylation/dephosphorylation events and the translocation of
cyclins as we have described during the death of cerebellar granule
neurons deprived of K+ (Padmanabhan et
al., 1999). In addition, our observations differ from that observed in
dying neurons of adult brains where upregulation of cell cycle markers
has been reported (McShea et al., 1997; Nagy et al., 1997; Vincent et
al., 1997; Busser et al., 1998). This may be caused by the
downregulation of cell cycle molecules that occur in more mature neurons.
Role of E2F/DP complexes in neuronal death evoked by
DNA damage
What are possible consequences of pRb/p107 phosphorylation and
loss of pRb and p107 protein? pRb binds E2F and negatively regulates
its transcriptional activity; when hyperphosphorylated by Cdk4/6, pRb
releases E2F, resulting in the derepression of E2F-responsive genes
(Chellappan et al., 1991). This raised the possibility that E2F plays a
causal role in the death of camptothecin-treated cortical neurons. E2F
members 1-5 are present in embryonic cultured cortical neurons
(D. S. Park, unpublished data). Of additional relevance,
overexpression of E2F in a variety of non-neuronal cells triggers
apoptotic death (Qin et al., 1994; Hiebert et al., 1995), and we have
observed that overexpression of E2F1 induces death of neurons (D. S. Park, D. Liu, and L. A. Greene, unpublished data). One means we
used to test the possible role of E2F in death was overexpression of
K11 Rb. This protein has been shown to suppress E2F-mediated
transcription (Brown et al., 1999). We observed that this construct
protected neurons from camptothecin-evoked death, consistent with a
role for E2F in the apoptotic mechanism.
As a second test of our hypothesis, we used Sindbis virus to express
several different DN constructs of DP1 (a member of a family of
obligate partners for E2F-transactivating activity) (Bandara et al.,
1993; Girling et al., 1993; Wu et al., 1996) in cultures of cortical
neurons exposed to camptothecin. These DN forms, but neither WT DP1 nor
a variety of control constructs, provided significant protection from
death evoked by camptothecin. Similar results were obtained with
sympathetic neurons exposed to UV irradiation. The findings provide
further support for a role of E2F/DP1 complexes in neuronal death
evoked by camptothecin and suggest that this extends to multiple
neuronal types and to additional paradigms of DNA damage.
It must be noted that there are alternative means by which DN DP1 may
provide neuronal protection. The DN versions of DP1 used in these
studies contain a deletion of the DNA-binding domain that is required
for mediating E2F activity and cell cycle progression in proliferating
cell systems (Wu et al., 1996). The apparent requirement for the
DNA-binding domain in death signaling suggests the importance of
transcriptionally mediated events. In support of this possibility,
neuronal death attributable to DNA damage requires transcription
(Morris and Geller, 1996; Park et al., 1998a). A recent report,
however, suggests that E2F may mediate death in at least certain
proliferating systems via a transcriptionally independent mechanism and
that this requires the E2F DNA-binding domain (Hsieh et al., 1997).
Accordingly, it is unclear whether transcriptional regulation mediated
by E2F/DP complexes is important in neuronal apoptosis. It is important
to note that it is unclear which E2F complexes may be inactivated by DN
DP1 expression in neurons. Curiously, we find that DN DP1 expression is
not exclusively nuclear. Wu et al. (1996) have suggested that DN DP1 is
able to inhibit the transcriptional activity of multiple E2F members
(1-5). Because several of the E2F members are known to also reside in the cytoplasm [for example, E2F4 (Verona et al., 1997)], it is possible that DN DP1 may act (partially or fully) in the cytoplasm to
inhibit death.
Finally, E2F and DP are also reported to interact physically with other
proteins including p53 both in vitro and in proliferating cells (O'Connor et al., 1995; Sorensen et al., 1996). The existence of
such interactions in neurons and/or their potential functions are
unknown. Accordingly, we cannot exclude the possibility that expression
of DN DP1 may protect neurons via a mechanism that includes p53 or
other proteins. In this regard, it has been reported that p53
expression is required for the death of neurons evoked by DNA damage
(Enokido et al., 1996; Johnson et al., 1998; Anderson and
Tolkovsky, 1999).
The findings presented here provide a plausible model as to why a
variety of inhibitors of the cell cycle are effective in rescuing
neurons from the death evoked by DNA damage. Our data suggest that a
post-translational mechanism is activated in camptothecin-treated cortical neurons that elevates Cdk4/6 activity and that this leads to
enhanced phosphorylation of pRb/p107. Phosphorylation of these proteins
in turn contributes to their loss by a mechanism requiring caspase
activity. Our findings that expression of K11 Rb and DN DP1 protects
neurons from DNA damage further suggest that loss of pRb and p107
permit dysregulated E2F transactivational activity that results in
synthesis of gene products that contribute to and are required for the
death process. Elements of additional pathways such as p53 (Enokido et
al., 1996; Johnson et al., 1998) and c-Jun/JNK (Estus et al., 1994; Ham
et al., 1995; Xia et al., 1995; Maroney et al., 1999) have been
implicated in neuronal death. It remains to be seen whether these act
in parallel, or intersect, with the pathway described here.
| |
FOOTNOTES |
Received Aug. 30, 1999; revised Feb. 9, 2000; accepted Feb. 17, 2000.
This work was supported in part by grants from the National Institutes
of Health-National Institute of Neurological Disorders and Stroke and
the Blanchette Rockefeller Foundation (L.A.G.), the National Institutes
of Health (H.M.G. and M.L.S.), the National Cancer Institute of
Canada with funds from the Canadian Cancer Society (R.B.), and
the Medical Research Council of Canada and Glaxo Wellcome (D.S.P.) We
would like to thank Jie Wu for technical support.
D.S.P. and E.J.M. contributed equally to this manuscript.
Correspondence should be addressed to Dr. David S. Park, Neuroscience
Research Institute, University of Ottawa, 451 Smyth Road, Ottawa,
Ontario, K1H 8M5, Canada. E-mail: dpark{at}uottawa.ca.
Dr. Morris's present address: Massachusetts General Hospital Cancer
Center, Laboratory of Molecular Oncology Building 149, 13th Street
Mailcode 149-7330, Charlestown, MA 02129.
| |
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ABSTRACT
INTRODUCTION
