(Received for publication, November 26, 1996, and in revised form, April 8, 1997)
From the To study the molecular mechanisms underlying
neuronal programmed cell death (PCD), we performed differential display
screening for genes, the expression of which was induced during PCD in
the sympathetic neuron culture model deprived of NGF. We cloned a gene
encoding a novel polypeptide (DP5) which consisted of 92 amino acids.
DP5 polypeptide had no homology with any other known protein and
contained no motif that would indicate its putative biochemical
functions. DP5 mRNA levels peaked at 15 h after nerve growth
factor withdrawal, concurrent with the time at which neurons were
committed to die. The induction of DP5 gene expression was blocked when
cell death was rescued by treatment with cycloheximide, KCl, or the
cyclic AMP analogue CPTcAMP. Overexpression of the full-length DP5 in
cultured sympathetic neurons was in itself sufficient to induce
apoptosis. These results suggest that DP5 plays a role in programmed
neuronal death.
Programmed cell death (PCD)1 is an
indispensable phenomenon for proper development of the nervous system.
Roughly half of all neurons produced by neurogenesis die during
development of the mammalian nervous system (1). Neuronal survival is
determined largely by neurotrophic factors such as NGF produced by
target cells, and neurons that do not obtain an adequate supply of
survival factors undergo apoptosis (2). Dissociated sympathetic neurons from rat superior cervical ganglia (SCG) are mainly used as an in
vitro model of neuronal PCD and have been characterized
extensively (3, 4). In this culture system, the majority of neurons die
after removal of NGF from the culture medium. The characteristics of
this neuronal death such as shrinkage of the neuronal soma with intact
organelles, nuclear condensation, fragmentation of the DNA into
oligonucleosomes, and blebbing of the plasma membrane are hallmarks of
apoptosis (5, 6). Neuronal death in this system can be prevented by
inhibitors of RNA or protein synthesis, suggesting that this phenomenon
is controlled by a genetic program (7).
In the search for constituents of the genetic program, several genes
have been identified using this in vitro model. Immediate early genes such as c-fos and c-jun and cell
cycle-related genes such as c-myb and cyclin D1
(8, 9) are these candidates. Especially cell cycle-related genes
stimulate the postmitotic neurons to attempt re-entry into the cell
cycle, and these comflicting growth-regulatory signals are thought to
cause neurons to undergo apoptosis. On the other hand, several
anti-apoptotic proteins such as Bcl-2 (10, 11), adenovirus E1B19k (12),
or cowpox virus CrmA gene products (13) have been reported.
Overexpression of the bcl-2 gene products in sympathetic neurons has
been shown to protect neurons from apoptosis (10). Anti-apoptotic
properties of Bcl-2 have been reported to prevent the loss of
mitochondrial function (14). CrmA gene products also prevent neuronal
death induced by NGF deprivation. This activity is attributed to
interleukin 1 Re-entry into the cell cycle and activation of interleukin
1 Sympathetic neurons from the superior cervical
ganglia of 1-day-old Sprague Dawley rats were isolated and cultured as
described by Ham et al. (17). Digested cells were preplated
to minimize the number of non-neuronal cells in uncoated 10-cm dishes
for 3 h. The resultant supernatant enriched in neurons was then
plated on 10-cm dishes coated with collagen (Koken, Tokyo, Japan) in DMEM containing 10% fetal calf serum (FCS), 2 mM
glutamine, 20 µM fluorodeoxyuridine, 20 µM
uridine, and 50 ng/ml 2.5 S NGF (Promega). The neurons were maintained
for 5-7 days in the presence of NGF before being subjected to
screening, Northern blotting, and RT-PCR analyses. NGF withdrawal was
carried out by changing the medium to DMEM/10% FCS lacking NGF and
containing anti-NGF antibodies (CIDtech Research Inc., Ontario, Canada)
diluted 1:1000.
PC12 cells were maintained in DMEM containing 5% FCS and 10%
heat-inactivated horse serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Neuronal PC12 cell cultures were prepared by treatment
with 50 ng/ml NGF in DMEM containing 5 µg/ml transferrin, 5 µg/ml
insulin, and 0.02 µM progesterone for 7-10 days.
Deprivation of NGF was performed as for SCG neurons.
Total RNA was isolated by
the acid guanidine thiocyanate/phenol/chloroform method (18) from SCG
neurons cultured for 15 h in medium containing NGF and those
cultured in medium from which NGF had been removed. Differential
display was carried out essentially as described previously (16).
cDNAs were synthesized from total RNA with reverse transcriptase
after DNaseI treatment, and PCR was performed using a ramdom primer
with the synthesized cDNA as a template. We used about one hundred
different random primers (12-mers, Common primer, Bex Inc.) for
screening. After separation by 5% polyacrylamide gel electrophoresis,
cDNA bands of interest were cut from the gel, reamplified using the
same primers from the eluted cDNA solution, and then cloned into
the pGEM-T vector (Promega).
A 630-bp DP5 cDNA fragment
(DP-PCR) obtained by differential display was used to screen 1 × 106 plaques from each of a newborn rat brain Uni-ZAP XR
cDNA library, an adult rat brain Total RNA was fractioned by
electrophoresis through 1.0% agarose/formaldehyde gels and transferred
onto Immobilon N membranes (Millipore). Filters were hybridized with
32P-labeled cDNA probe generated from the DP5 cDNA
DP-PCR, with a specific activity between 5 × 108 and
3 × 109 cpm/µg DNA by the random hexamer procedure
(19). After washing in 2 × SSC, 0.1% SDS and 0.1 × SSC,
0.1% SDS, filters were dried and autoradiographed.
Aliquots of 3 µg of total RNA purified
from preplated SCG cultures were reverse-transcribed using 300 units of
Moloney murine leukemia virus reverse transcriptase (Life Technologies,
Inc.) in a 60-µl reaction mixture in the presence of 2.5 µM oligo(dT) primer and 20 µM dNTP mixture
for 60 min at 37 °C. For PCR amplification, specific oligonucleotide
primer pairs (0.5 µM each) were incubated with 1 µl of
cDNA, 1 unit of Taq polymerase, 1 × Taq buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2), 10 µM dNTP mixture, and 10 µCi of [ Sf21 insect cells were infected with
recombinant baculovirus carrying glutathione S-transferase
(GST)-DP5. Recombinant fusion proteins extracted with Triton X-100 were
purified by chromatography on glutathione-agarose (Pharmacia Biotech
Inc.). Typical yield of fusion protein was 1 mg per 2 × 108 infected Sf21 cells. Antibodies were raised against the
recombinant GST-DP5 fusion protein. Immunization of rabbits and
screening of antisera were performed as described previously (20).
For detection of DP5 polypeptide, 1 × 106 SCG neurons
were plated per 10-cm dish and incubated for 7 days in the presence of NGF. One dish was used per immunoprecipitation. Neurons were harvested and then lysed in 1 ml of buffer containing 10 mM Tris-HCl
(pH 7.4), 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 10 µg/ml aprotinin
(Sigma). Lysates were centrifuged at 13,000 rpm for 5 min to remove
large cellular debris. For each immunoprecipitation, 3 µg of the
anti-GST-DP5 fusion antibody was used. Samples were incubated for
1 h at 4 °C on a rocker with the antibody. Anti-rabbit IgG
agarose (Sigma, 20 µl) was added to each sample followed by another
1-h incubation at 4 °C on a rocker. The beads were then washed five
times in the lysis buffer to reduce nonspecific binding. On the last
wash, all buffer was removed, and reducing sample buffer was added to
each reaction. Samples were boiled and loaded onto a 4-20% gradient
SDS-polyacrylamide gel. After electrophoresis, gels were transferred to
Immobilon P (Millipore). Blots were preblocked in phosphate-buffered
saline (PBS) containing 5% nonfat milk, and washes were performed
using PBS containing 0.1% Tween 20 (PBS-T). Primary antibodies were used at 0.2-0.5% (v/v), and detection was performed with 0.1% alkaline phosphatase-conjugated goat anti-rabbit IgG (Boehringer Mannheim) in PBS-T with 5% nonfat milk by the alkaline phosphatase method.
The DP5 cDNA DP-7W was subcloned
in both sense and antisense orientation into the XbaI site
of pEF-BOS (21, pEFDP5 and pEFantiDP, respectively). For the control
vectors, the lacZ gene (3.5-kbp cDNA containing the open
reading frame of Microinjection was carried out using a
Narishige micromanipulator. Microinjection needles were pulled from
glass capillaries using a vertical electrode puller and loaded using
Eppendorf microloaders. DNA was injected directly into the nucleus.
Each expression vector, pEFDP5, pEFantiDP, and pEFlacZ, was injected in
0.5 × PBS at a concentration of 0.05-0.2 mg/ml. Approximately
80-90% of SCG neurons survived after microinjection.
For analysis of the effects of expression vectors on neuronal cell
death, neurons were plated at a density of 100 per measure (3 × 3 mm) and used 5-7 days after plating. Fluorescein
isothiocyanate-labeled goat anti-mouse IgG (The Jackson Laboratory) was
used to mark the injected cells in these experiments and was added to
the injection mixture at a final concentration of 0.5 mg/ml. The
neurons were maintained in the presence of NGF, and the numbers of
living cells were counted based on morphological criteria and trypan
blue staining at various time points after microinjection. For X-gal
staining of neurons expressing TUNEL was carried out essentially as
described previously (22). Cells were fixed for 30 min in 4%
paraformaldehyde at room temparature. They were then rinsed three times
in PBS and incubated in permeabilization solution (0.1% Triton X-100,
0.1% sodium citrate) for 2 min on ice. After washing twice, in
situ labeling of apoptosis-induced DNA strand breaks was performed
using an In situ Cell Death Detection Kit, fluorescein
(Boehringer Mannheim). The cells were analyzed directly under a
fluorescence microscope.
Previously, the commitment point for cell death in the
culture model, as defined by the ability of half of the neurons to be
rescued from death by readdition of NGF or addition of cycloheximide (CHX), has been shown to occur after approximately 15-17 h (23, 24),
and this was confirmed by our results (data not shown). Sufficient
quantities of "death gene" or "death-related gene" products and
their mRNA would have accumulated by this time. Therefore, we
collected mRNAs from SCG cultures 15 h after NGF withdrawal. The control mRNAs were isolated from SCG neurons which were
cultured in medium containing NGF. These mRNAs were
reverse-transcribed and screened for genes induced during neuronal PCD
by the differential display technique (see "Experimental
Procedures"). Although most of the bands observed in this screening
showed the same patterns in both control neurons and those in which PCD
was induced, we isolated one clone (DP5), the expression of which was
strongly induced during cell death, and the length of the cDNA
fragment amplified by PCR was 630 bp (designated as clone DP-PCR, Fig. 1). The nucleotide sequence of the cDNA fragment
showed no homology with those of known genes registered in the EMBL,
GenBankTM, and DDBJ data bases. By Northern blot analysis,
a cDNA probe synthesized from 630 bp of the DP5 cDNA DP-PCR was
shown to hybridize with a single mRNA species of about 5.5 kb, and
expression of DP5 mRNA was specific to the neurons in which PCD was
induced (Fig. 2A).
The sequence of events that occur following removal of NGF from
differentiated PC12 cells is very similar to that in sympathetic neurons following removal of NGF (3), and the commitment point in the
neuronal PC12 cell model system is considered to be 12-15 h after NGF
deprivation (25). To show that induction of DP5 is not specific to SCG
neurons, we examined expression of the DP5 gene during PCD of neuronal
PC12 cells. As shown in Fig. 2B, PC12 cells cultured in
medium containing NGF expressed DP5 mRNA only faintly, whereas
those deprived of NGF showed elevation of DP5 mRNA expression. The
level of expression of DP5 mRNA was higher 15 h after removal
of NGF, and this time was consistent with the commitment point for PCD
in this model.
We next examined tissue distribution of DP5 mRNA. Among the various
adult rat tissues examined, only the brain showed a hybridizing band
for the DP5 mRNA (Fig. 2C). No signals were detected in
other tissues examined even if the membrane was exposed to film for 7 days. Therefore, it appears that expression of the DP5 gene could be
highly specific to the nervous system.
We then isolated and
determined the full nucleotide sequence of DP5 cDNA. The cDNA
consists of 5253 nucleotides, and many stop codons were found in this
nucleotide sequence. There were three possible open reading frames
(ORF) in all three frames. In all cases, these ORFs encoded small
polypeptides which consisted of about 100 amino acids. To determine the
ORF of DP5, we isolated a cDNA of mouse DP5 homolog from newborn
mouse brain and characterized it. Comparison of a 279-bp segment of an
ORF at its 5
To demonstrate that the DP5 polypeptide is indeed expressed
during neuronal death, a rabbit polyclonal anti-GST-DP5 fusion protein
antibody was raised against proteins produced in a baculovirus expression system, and immunoprecipitation followed by Western blotting
was carried out. An immunoreactive band of approximately 10 kDa was
detected in Sf21 insect cell homogenates expressing a 1.5-kbp cDNA
fragment containing the ORF (clone DP-7W, Fig. 4). This
immunoreactive band was also observed in SCG neurons 24 h after
NGF deprivation, but not in those of control neurons.
To
examine the temporal changes in levels of DP5 mRNA during PCD, we
reverse-transcribed mRNAs isolated from SCG cultures before and at
varying times after NGF withdrawal and analyzed proportions of DP5
cDNAs using RT-PCR (Fig. 5A). We also
analyzed the expression patterns of control cellular marker genes
including the neuronal gene neurofilament-M (NF) and the glial and
Schwann cell marker S-100
We next examined the changes in DP5 mRNA expression on treatment of
SCG neurons with various agents which prevent neuronal PCD,
i.e. CHX, KCl (26, 27), and the cyclic AMP analogue CPTcAMP (24, 28). To test the abilities of various agents to promote survival,
SCG neurons deprived of NGF were cultured for 24 h with or without
various agents, and the number of survived neurons were counted.
Without additives, approximately 90% of the cells died, while good
survival was promoted in the presence of CHX, KCl, or CPTcAMP (Fig.
5B). After neuronal cultures were deprived of NGF for
15 h in the presence or absence of various agents, total RNA was
extracted and analyzed for changes in DP5 gene expression by RT-PCR.
Levels of products amplified by DP5-specific primers were markedly
decreased in all cases treated with cell death blockers (Fig.
5C). Quantification of the DP5 signals showed that the
expression levels of DP5 in SCG neurons treated with various agents
were approximately 5-30% of those in control neurons (Fig.
5D). The degrees of cell death prevention corresponded well
with the reduction of DP5 gene expression by treatment with these
agents.
Having demonstrated
that expression of DP5 increases after NGF withdrawal and that DP5 is
closely associated with neuronal PCD, it was of interest to determine
whether overexpression of DP5 alone is sufficient to cause the death of
SCG neurons. We microinjected neurons with a full-length DP5
polypeptide expression vector (pEFDP5) and with antisense DP5 vector
(pEFantiDP) or a
To verify that this type of cell death is caused by apoptosis but not
by nonspecific stress or toxicity of overproduction of DP5 polypeptide,
we coinjected expression plasmids of bcl-2 gene together
with the DP5 gene into the SCG neurons. Most of the injected neurons
did not die 24 h after microinjection of bcl-2 and DP5
expression vectors (Fig. 8). This suggested that Bcl-2
can prevent cell death induced by DP5 and that this type of cell death
is not simply caused by toxicity of overproduction of DP5 polypeptide.
This notion was also supported by the facts that neither
PCD in a variety of systems requires de novo gene and
protein expression, evidenced by the ability of transcriptional or
translational inhibitors to block PCD (3, 29-33). This indicates that
cell death genes and proteins which control the cell death program are
induced during PCD. We isolated one clone, DP5, strongly induced during
PCD of cultured SCG neurons. The putative ORF of DP5 gene was very
small and was located at the 5 The induction of DP5 mRNA during neuronal PCD was demonstrated
using RT-PCR and Northern blotting. During PCD, DP5 mRNA began to
accumulate 5 h after NGF withdrawal and reached maximal levels at
15 h, with a subsequent gradual decrease by 25 h. The peak of
DP5 expression correlated well with the commitment point for cell death
(15-17 h) as determined by CHX rescue after NGF deprivation (23, 24).
DP5 was not induced immediately early, but many hours after the initial
stimulus of NGF removal, suggesting that DP5 was induced in response to
intracellular events. Indeed, the induction of DP5 expression was
blocked by CHX. This finding suggests that DP5 could be a part of a
cellular genetic program that required ongoing protein synthesis.
Cell death was prevented by treatment with high K+, which
depolarized neurons and raised intracellular Ca2+ levels
(26, 27), or by treatment with cAMP analogue CPTcAMP (23, 28). In these
cases, the expression of DP5 was markedly suppressed. The degrees of
suppression of DP5 expression corresponded with those of cell death
blockade by these agents. The mechanisms by which these agents prevent
the death program remain unclear. However, the results suggest that DP5
expression is closely associated with proceedings of neuronal death,
and DP5 is likely to be a constituent of the death program in SCG
neurons.
Neurons microinjected with pEFDP5 to overexpress DP5 gene products
underwent apoptosis even in the presence of NGF. Cells into which DP5
was introduced showed typical morphological characteristics of
apoptosis such as shrinkage of the cell body, blebbing of the plasma
membrane, and DNA fragmentation as evidenced by TUNEL staining. Deckwerth and Johnson (24) reported morphological changes of sympathetic neurons after removal of NGF. In this paper, 19 h after NGF withdrawal, atrophy was first detectable in half of the
neuronal somas, and the plasma membrane lost its smooth appearance and
its cellular outline became increasingly irregular. These observations
corresponded with morphological changes of neurons injected with DP5
expression vectors, suggesting that the cells into which the DP5
constructs were introduced could be activated death pathway and die in
an apoptotic fashion. In contrast, cells injected with antisense DP5,
lacZ, In conclusion, the present study strongly suggests that DP5 is likely
to be one of the constituents of the genetic program of neuronal PCD.
Whether DP5 is required for this phenomenon or not is open for further
analysis. Experiments to test this question are currently in progress,
using gene knockout technologies. Furthermore, identification of the
protein(s) which interact(s) with the DP5 polypeptide and transcription
factors which regulate DP5 gene expression are necessary to elucidate
the putative DP5-related cell death cascade.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D83697. We thank Prof. Y. Tsujimoto (Division of
Molecular Genetics, Osaka University Medical School) for helpful
discussions. We are grateful to Dr. S. Nagata (Osaka Bioscience
Institute, Japan) for providing the expression vector pEF-BOS, and we
would like to thank Prof. Y. Yoneda and Dr. Y. Matsuoka (Anatomy of
Cell Biology, Osaka University Medical School) for advice on
microinjection.
When this manuscript was accepted for publication,
we noticed a paper that described cloning of a gene,
harakiri (Hrk), which was significantly
homologous with DP5 (35). Judging from the differences of sizes of the
transcripts and expression patterns, DP5 seems not to be a rat
homologue of Hrk, but to constitute a gene family with
Hrk. Both proteins can induce apoptosis when overexpressed
in the cultured cells. We do not know whether DP5 can specifically
interact with BCL-2 and BCL-Xl as Hrk does. This aspect is
now under investigation.
Department of Molecular Neurobiology,
Department of Cell Science,
Institute of Biomedical Sciences, Fukushima Medical College, Fukushima
960-12, Japan
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
Addendum
REFERENCES
-converting enzyme protease inhibition (13,
15).
-converting enzyme-like proteases are closely associated with neuronal PCD, but the molecular events or cascades underlying neuronal
death regulated by the genetic program still remain unclear. To
elucidate the mechanisms, it is crucial to list the molecules involved
in this process and to examine the relationships between them. For this
purpose, using the differential display technique (16), we screened for
genes, the expression of which is induced during neuronal PCD, and
identified one candidate, named DP5. Here we report its structure,
expression patterns, and putative biological functions.
Cell Culture
ZAPII cDNA library, and a
newborn mouse brain
ZAPII cDNA library (Stratagene). Standard
phage screening techniques were employed. Several rounds of screening
were performed using 3
- and 5
-regions of cDNA fragments obtained
by the first screening as probes to obtain overlapping clones almost
covering the full-length DP5 cDNA.
-32P]dCTP in a 20-µl reaction mixture.
Oligonucleotides used for amplification of DP5 cDNA were DP5-5
,
5
-AGACCCAGCCCGGACCGAGCAA-3
, 347-368; DP5-3
,
5
-ATAGCACTGAGGTGGCTATC-3
, 516-535. Typical cycle parameters were 1 min at 94 °C, 1 min at 60 °C, and 30 s at 72 °C for
22-24 cycles followed by 72 °C for 5 min. 10-µl aliquots of each
reaction mixture were electrophoresed through 5% polyacrylamide gels,
and the dried gels were subjected to autoradiography and quantitative
analysis (Scanning Imager, Molecular Dynamics). Control experiments
were performed to determine the range of PCR cycles over which
amplification efficiency remained constant. The identity of each PCR
product was confirmed by subcloning the amplified cDNAs into pGEM-T
vector (Promega) and sequencing.
-galactosidase derived from pCH110,
CLONTECH) were cloned into pEF-BOS (pEFlacZ). The rat bcl-2 cDNA was cloned by PCR using RNA from rat brain and was
also cloned into the XbaI site of pEF-BOS.
-galactosidase, cells were fixed in
1% glutaraldehyde for 3 min and stained with X-gal solution (100 mM sodium phosphate buffer, pH 7.2, 10 mM KCl,
1 mM MgCl2, 3 mM K3Fe(CN)6, 3 mM
K4Fe(CN)6, 0.1% Triton X-100, and 0.1% X-gal) at 37 °C.
Identification of DP5, a Gene Induced during Neuronal
PCD
Fig. 1.
Differential display. Autoradiography of
amplified 35S-labeled PCR products (electrophoresis in a
5% polyacrylamide gel) using an arbitrary primer
(5-AACC(A/G)AA(A/G)TCNG-3
). C, normal sympathetic neurons;
D, neurons 15 h after NGF deprivation. #1 and #2 indicate different sources of neurons cultured on
separate days, respectively. The arrow shows bands of
DP5.
[View Larger Version of this Image (59K GIF file)]
Fig. 2.
Northern blot analysis of DP5 mRNA
expression. A, DP5 mRNA expression in SCG neurons
15 h after NGF deprivation. The 32P-labeled DP5
cDNA probe hybridized to an RNA species of approximately 5.5 kb.
Total RNA isolated from control neurons or from neurons in which PCD
was induced was loaded in each lane. Ethidium bromide-stained ribosomal
RNAs (18 S) indicate that the amounts of total RNAs were nearly
equivalent in each lane. B, changes of DP5 mRNA
expression in neuronal PC12 cells induced to undergo apoptosis by
removal of NGF. Total RNAs were purified from the PC12 cells at the
specified times (0, 15, 24 h) after NGF deprivation. C,
the expression of DP5 in various tissues of an 8-week-old male Sprague
Dawley rat. B, brain; Sp, spleen; Ty,
thymus; Te, testis; H, heart; I,
intestine; Sk, skeletal muscle. The signal of DP5 mRNA
seems greatly intense in brain, but this filter was autoradiographed
for 7 days to confirm that DP5 mRNA is not expressed in other
tissues. Originally, the signal of DP5 is very faint. 15-µg aliquots
of total RNA were loaded in each lane.
[View Larger Version of this Image (36K GIF file)]
end, which has the 5
-most ATG among the cDNA clones
isolated, from rat and mouse revealed only one nucleotide change, and
this change did not alter the predicted amino acid sequence of the ORF
(Fig. 3B). In contrast, the other regions of
mouse DP5 cDNA including another two possible ORFs showed 84%
homology with those of rat cDNA. These observations suggest that
there is selective pressure to conserve this 279-bp segment as a
protein coding region, and DP5 encodes a polypeptide of 92 amino acids.
Data base analysis showed that DP5 had no homology with any other known
protein and contained no motif that would indicate its putative
biochemical functions. Hydrophobicity analysis indicated that DP5 does
not contain a signal sequence or any transmembrane regions.
Fig. 3.
Structure of DP5 cDNA, predicted amino
acid sequence. A, the overall structure of the 5253-bp DP5
cDNA and the positions of selected restriction sites are indicated
by the arrows. DP5 cDNA clones obtained by differential
display (DP-PCR) and used for introduction into neurons on
microinjection (DP-7W) are also shown. The size
bar represents 300 bp. B, predicted amino acid sequence
of DP5. The nucleotide sequence of the open reading frame is shown on
the top line. The second line shows the
homologous sequence from mouse. We found 1-base changes within 279 bp
of the ORF, none of which changed the amino acid sequence of this ORF.
As shown below the DNA sequence, this gene gives rise to a peptide of
92 amino acids. The full cDNA sequence can be obtained from
GenBankTM (accession number D83697).
[View Larger Version of this Image (22K GIF file)]
Fig. 4.
Immunoblot analysis of DP5 polypeptide
expression during neuronal PCD. Following immunoprecipitation with
anti-GST-DP5 fusion protein antibody, homogenates from Sf21 cells
overexpressing DP5 polypeptide and cell extracts from the control SCG
neurons or from neurons 24 h after withdrawal of NGF were
separated by SDS-PAGE and then transferred onto a polyvinylidene
difluoride membranes (Millipore). The filters were incubated with the
antibodies and then stained by the alkaline phosphatase method. Sizes
of standard proteins are shown in kilodaltons. The arrow
shows the immunoreactive band of DP5. * and ** indicate nonspecific
bands.
[View Larger Version of this Image (42K GIF file)]
. Expression of S-100
did not
significantly change over the 25-h period of NGF withdrawal. PCR
products of NF diminished over the course of PCD. Thus, mRNA levels
for neuronal and non-neuronal genes decreased or did not change during
PCD. The DP5 expression pattern during PCD was in contrast with the patterns of expression of the control markers described above. A
relatively low level of DP5 expression was detected before NGF withdrawal. This signal increased at 5 h and was maximal at
15 h after removal of NGF with a subsequent reduction after this time point. The temporal changes in DP5 mRNA expression during PCD
matched the expression pattern predicted for putative cell death genes
or death-related genes, i.e. the peak of DP5 expression occurred concurrently with the commitment point for PCD as determined by CHX rescue after NGF withdrawal.
Fig. 5.
RT-PCR analysis of DP5 expression.
A, time course of DP5 mRNA expression during cell death of SCG
neurons. Primary cultures (10-cm dishes) were maintained with NGF for
5-7 days and then deprived of NGF for the indicated intervals. Total
RNA was isolated, and 3-µg aliquots of RNAs were reverse-transcribed. Then 1/60 volume of cDNA solution was used to examine expression of
the following genes by PCR (number of PCR cycles in parentheses): DP5
(24 cycles), neurofilament-M (NF, 22 cycles), and S-100
(24 cycles). The amplified DNAs were separated on 5% polyacrylamide gels and visualized by autoradiography. The identity of each PCR product was confirmed by DNA sequencing. B, neuronal death
prevented by various agents. After SCG cultures were deprived of NGF
for 24 h in the presence or absence of 1 µg/ml CHX, 35 mM KCl, or 400 µM CPTcAMP, the number of
dying cells was counted. The data (mean ± SD) shown were the
percentages of dead cells. Data were collected from at least five
independent experiments. C, using RT-PCR assay, analysis of
changes of DP5 expression 15 h after NGF deprivation in the cases
treated with various cell death blockers. NF was used as an internal
control. D, quantification of changes of DP5 expression
suppressed by cell death blockers. Changes in the levels of DP5 were
quantified by Scanning Imager (Molecular Dynamics) analysis of
polyacrylamide gels such as that shown in C. Changes were
determined relative to the DP5 expression levels in the control with
deprivation of NGF and represent the means from three analyses.
[View Larger Version of this Image (42K GIF file)]
-galactosidase expression vector (pEFlacZ) as a
negative control. The neurons were maintained in the presence of NGF,
and the numbers of living cells were counted at various time points
after injection. Most cells injected with pEFlacZ and pEFantiDP at a
concentration of 0.1 mg/ml survived at 24 h after injection. In
contrast, about 70% of neurons injected with pEFDP5 at an equivalent
concentration died by this time point (Fig. 6). The cell
death caused by introduction of DP5 gene begun at 3 h after
injection and surviving cell number was rapidly decreased at 6 h.
The dying cells overexpressing DP5 showed morphological characteristics
of apoptosis such as shrinkage of the cell body, pyknotic nuclei, and
blebbing of the plasma membrane (Fig. 7,
A-D). TUNEL (TdT-mediated dUTP nick
end-labeling) staining revealed positive reactivity in the nuclei of
shrinking cells injected with pEFDP5 (Fig. 7F), but those of
cells injected with pEFlacZ and pEFantiDP were negative for TUNEL
staining (data not shown). These results suggested that cell death
induced by DP5 involved DNA fragmentaion.
Fig. 6.
Effects of DP5 overexpression on the survival
of SCG neurons in the presence of NGF. Neurons were microinjected
with expression vectors containing sense DP5 cDNA DP-7W
(sense), containing DP-7W in antisense orientation
(antisense), or containing the lacZ gene
(lacZ). Changes in the survival of neurons at the specified time points in the presence of NGF were determined relative to the
number of neurons 1 h after microinjection. Results are presented as the mean percentages of surviving cells ± SD observed in 6 experiments (1000 microinjected for each vector).
[View Larger Version of this Image (17K GIF file)]
Fig. 7.
Morphological changes in neurons injected
with DP5 expression vector. The injected neurons were cultured for
6 h in the presence of NGF. Phase-contrast photomicrographs of
neurons injected with pEFantiDP (A) and pEFDP5
(B). Neurons injected with DP5 expression vector showed
apoptotic changes such as shrinkage of the cell body and blebbing of
the plasma membrane. Bar = 40 µm. C and
D, X-gal staining of neurons coinjected with pEFantiDP and
pEFlacZ (C) and pEFDP5 and pEFlacZ (D). Apoptotic
neurons coinjected with pEFDP5 and pEFlacZ showed positive signal for X-gal staining. Bar = 40 µm. E and
F, TUNEL staining of neurons expressing DP5. The
DP5-injected neurons were cultured for 6 h in the presence of NGF
and then stained by the TUNEL method. E, phase-contrast
image; F, TUNEL staining. Shrinking cells showed positive
reaction for TUNEL staining (arrows). Bar = 20 µm.
[View Larger Version of this Image (147K GIF file)]
-actin nor
GST expression vectors killed SCG neurons (data not shown).
Fig. 8.
Bcl-2 prevents cell death induced by
DP5. Neurons were co-injected with pEFDP5 and Bcl-2 expression
plasmids at 0.02 mg/ml and cultured for 24 h in the presence of
NGF. The percentages of neuronal survival were determined relative to
the number of neurons 1 h after microinjection. The results shown
are means ± SD of at least 5 independent experiments (in each
experiment, at least 100 neurons were injected with each construct; **,
p < 0.01 versus percentages from the
DP5-injected control.)
[View Larger Version of this Image (62K GIF file)]
end of the 5.3-kbp DP5 cDNA.
Although this was not a usual position of ORF, we consider it valid for
the following two reasons. 1) The mouse DP5 homolog shows a very high
degree of conservation of the nucleotide sequence within this ORF. 2)
In Western blotting analysis, the polyclonal DP5 antibody reacted with
a 10-kDa band in SCG neurons undergoing PCD, which is consistent with
the size of the protein predicted from ORF. This gene and gene product
showed no homology with any other known genes or proteins and contained
no motif that may indicate a biochemical function. DP5, therefore, may
represent the prototype of a novel type of cell death gene. Another
unique feature of DP5 gene is that it is expressed only in the brain in
adult rats, and no expression was detected in other tissues. If the DP5
gene is also closely associated with the death program in
vivo as shown in the in vitro study, cell death
cascades in the nervous system, which accompany induction of DP5, may
differ from those of other tissues. In support for this hypothesis, the expression of DP5 was not detected during cell death of thymocytes induced by glucocorticoid (data not shown). Whether DP5 overexpression can induce cell death in other types of cells is an interesting point
and is now under investigation in our laboratories.
-actin, or GST expression vectors showed no
morphological changes and did not die. In addition, most of the neurons
co-injected with bcl-2 and DP5 did not die. Taking these
results into account, it is unlikely that the cell death was caused by
nonspecific toxicity of DP5 overproduction. Injecting DP5 kills neurons
rapidly even in the presence of NGF. This suggests that DP5 may be
having an effect late in the death processes unlike most other
"killer genes" such as c-jun and myc. As
described above, the induction of DP5 expression was inhibited by CHX
or some other cell death blocker, i.e. it is required for ongoing protein synthesis and may be needed for execution of cell death. However, we cannot unveil the molecular cascades of neuronal apoptosis induced by DP5 in the present sudy. There are several precedents which can induce apoptosis of neuronal cells, such as Bak
(34) and c-Jun (17). Whether DP5 interacts or associates with these
proteins must be analyzed further to understand the mechanisms of
DP5-induced cell death.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Molecular
Neurobiology, "TANABE," Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. Tel.: 81-6-879-3646; Fax: 81-6-879-3648; E-mail: imaizumi{at}brain.med.osaka-u.ac.jp.
1
The abbreviations used are: PCD, programmed cell
death; SCG, superior cervical ganglia; FCS, fetal calf serum; CHX,
cycloheximide; ORF, open reading frame; GST, glutathione
S-transferase; NF, neurofilament-M; TUNEL, TdT-mediated dUTP
nick end-labeling; PBS, phosphate-buffered saline; NGF, nerve growth
factor; DMEM, Dulbecco's modified Eagle's medium; RT-PCR, reverse
transcription-polymerase chain reaction; bp, base pair(s); kbp,
kilobase pair(s); kb, kilobase(s); X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside; CPTcAMP, cyclic 8-(4-chlorophenylthio)adenosine 3
:5
-monophosphate.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.