From the Neurobiology Unit, Institut d'Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Científicas, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Rosselló 161, 08036 Barcelona, Spain
Received for publication, August 31, 2000
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ABSTRACT |
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Mature cerebellar granule cells in culture die by
a process that requires new RNA and protein synthesis when deprived of
depolarizing concentrations of potassium. We investigated gene
expression during the early phase of the cell death program evoked by
potassium deprivation. Using a differential gene display technique, we
isolated a cDNA that was increased by potassium deprivation. This
cDNA was homologous to the 3' mRNA end of neuronal pentraxin 1 (NP1), a gene encoding a secreted glycoprotein whose expression is
restricted to the nervous system. Reverse-Northern and Northern blot
analyses confirmed that treatment with low potassium induces
overexpression of NP1 mRNA, with a subsequent increase in NP1
protein levels. Time-course studies indicated that overexpression of
NP1 protein reaches a maximum after 4 h of exposure to potassium
deprivation and 4 h before significant cell death. Incubation of
cerebellar granule cells with an antisense oligodeoxyribonucleotide
directed against NP1 mRNA reduced low potassium-evoked NP1 protein
levels by 60% and attenuated neuronal death by 50%, whereas
incubation with the corresponding sense oligodeoxyribonucleotide was
ineffective. Furthermore, acute treatment with lithium significantly
inhibited both overexpression of NP1 and cell death evoked by low
potassium. These results indicate that NP1 is part of the gene
expression program of apoptotic cell death activated by nondepolarizing
culture conditions in cerebellar granule cells.
In the central nervous system, during normal embryonic
development, the number of neurons is adjusted by activation of a
built-in gene expression program that kills unnecessary cells in a
process described as programmed cell death (1). This process determines the size and shape of the vertebrate nervous system (2), and the cell
death that results from it exhibits the morphological features of
apoptosis (3). Apoptotic death is characterized by cell shrinkage,
nuclear condensation, DNA fragmentation at internucleosomal sites, and
degeneration of cell membrane-bound particles that are phagocytized by
macrophages without an inflammatory response. Recent evidence indicates
that gene expression-dependent apoptosis is also involved
in the pathological death of mature neurons observed in various
neurodegenerative disorders, such as Alzheimer's disease, or following
brain ischemia or traumatic injury of the central nervous system
(4-8). Nonetheless, the mechanisms involved in gene
expression-dependent apoptotic death of mature neurons are
not well characterized.
One of the most documented in vitro models to investigate
gene expression-dependent apoptosis in mature neuronal
cells is death induced by nondepolarizing culture conditions (9-11).
Cerebellar granule cells require depolarizing concentrations of
potassium (25-30 mM) to be maintained in culture (12).
When these cells are mature, reduction of the extracellular
concentration of potassium produces a cell death that is
morphologically apoptotic. This cell death evoked by low potassium is
associated with DNA fragmentation and requires both new RNA and protein
synthesis (9), because addition of protein or RNA synthesis inhibitors
within the first 4 h of exposure to potassium deprivation prevents
cell death and results in a complete recovery of the damaged DNA (9,
13-15). Likewise, replacement of high concentrations of potassium
within 4 h after treatment with low potassium results in no cell
loss. However, the activation of the cell death program becomes
irreversible in ~50% of cerebellar granule cells after 6 h of
exposure to low potassium (14).
In neurons, apoptotic death may be mediated by posttranslational
mechanisms as well as by "de novo" expression of death
genes. However, it has been proposed that the mechanisms involved in low potassium-mediated apoptosis of granule neurons are similar to
those operating in neuronal death during development or following blockade of neuronal activity, which require new mRNA and protein synthesis (9). Consequently, strategies directed to identify genes
whose expression is increased before neuronal cells reach the
commitment to die may help to identify new targets for neuroprotection. Based on this assumption, we have investigated gene expression during
the early phase of the cell death program.
Overexpression of several genes has been associated with cerebellar
granule cell death induced by low potassium. Thus,
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)1 overexpression has
been shown to be involved in the initiation of the apoptotic process
evoked by low potassium (16, 17). Other genes whose overexpression has
been found to mediate apoptosis by potassium deprivation include those
for the ICE-related protease CPP32 (18), c-Jun (10), Egr-1 (19), and
the transcription factor of cyclin-dependent kinases E2F-1
(20). However, the identification of other genes of the neuronal death
program is necessary to understand the relationship among the different
biochemical mechanisms of the apoptotic signal pathway activated by
reduction of synaptic activity.
Using a differential gene display technique (21), we have investigated
gene expression evoked by low potassium in cerebellar granule cells. We
now report that treatment with low potassium induces the overexpression
of neuronal pentraxin 1 (NP1), a gene that was originally identified
and isolated as a rat protein that mediates the
calcium-dependent uptake of the snake venom toxin, taipoxin
(22). NP1 encodes a glycoprotein of an apparent molecular mass of ~50
kDa that is predicted to be secreted and whose expression is restricted
to the nervous system (22). Our studies demonstrate that antisense
oligodeoxyribonucleotides against NP1 inhibit low potassium-induced
cell death. Thus, the present results provide evidence of a new
function for NP1 and indicate that NP1 is part of the gene program of
apoptotic death in cerebellar granule cells kept under nondepolarizing
culture conditions.
Cell Culture--
Primary cultures of cerebellar granule neurons
were prepared from 7-day postnatal Harlan Sprague-Dawley rat pups
(Harlan) as described previously (23, 24). Procedures involving animals and their care were approved by the ethics committee of the University of Barcelona and conducted in conformity with institutional guidelines that are in compliance with national (Generalitat de Catalunya decree
214/1997, DOGC 2450) and international (Guide for
the Care and Use
of Laboratory Animals, National
Institutes of Health publication 85-23, 1985) laws and policies. Cells
were dissociated in the presence of trypsin and DNase I, and plated in
dishes coated with poly-L-lysine (100 µg/ml). Granule
cells were seeded at a density of 3 × 105
cells/cm2 in basal Eagle's medium supplemented with 10%
fetal bovine serum (heat-inactivated), 0,1 mg/ml gentamicin, 2 mM L-glutamine, and 25 mM KCl.
Cerebellar granule cell cultures were kept at 37 °C in a humidified
incubator with 5% CO2, 95% air and remained undisturbed until experiments were performed. The replication of non-neuronal cells
was prevented by addition of 10 µM
cytosine-D-arabinofuranoside to the culture medium 24 h after plating. Cells were used for experiments after 8 days in
culture (8DIV).
Induction of Neuronal Death by Low Potassium--
Previous
studies have shown that cerebellar granule cells maintained in medium
supplemented with 25 mM K+ undergo apoptotic
death when switched to 5 mM K+ (9). In
addition, exposure of cerebellar granule cells to fresh
serum-containing medium triggers excitotoxicity (25). This
neurotoxicity does not occur if conditioned medium is used (25). Thus,
after 8 days in culture, the medium in which cerebellar granule cells
had grown, referred to as conditioned medium
(Sc+K+), was replaced with one of
the following media: fresh unconditioned serum-free medium supplemented
with 25 mM potassium (S
Treatments with lithium and antisense oligodeoxyribonucleotides (ODNs)
were performed at 8DIV immediately after the replacement of the media
described above. LiCl was added to the cultures at a concentration of 5 mM. In pilot experiments, we found that the optimal
concentration of ODNs was 10 µM.
Oligodeoxyribonucleotide Synthesis--
A 21-base-long
phosphorothioated antisense ODN against the NP1 mRNA and its
corresponding sense ODN were obtained from Roche Molecular
Biochemicals. The sequences were
5'-GCGTGCGGCGCGGCCGGCCAG-3' for the NP1
antisense ODN (NP1AS) and
5'-CTGGCCGGCCGCGCCGCACGC-3' for the
corresponding sense ODN (NP1S). The phosphorothioated nucleotides are
underlined. The NP1 antisense ODN sequence corresponds to nucleotides
4-24, which immediately follow the first initiation codon of the
coding sequence of the NP1 cDNA.
Determination of Cell Death--
Cell death was assessed using
propidium iodide (PI) staining. PI is excluded by the plasma membrane
of viable cells. Injury to the cytoplasmic membrane allows the entry of
PI, which, by interacting with nuclear DNA, yields a bright red
fluorescence. In time-course experiments, PI fluorescence was measured
in 24-well plates using a CytoFluor 2350 scanner (Millipore, Barcelona,
Spain) with 530 nm (25-nm band pass) excitation and 645 nm (40-nm band pass) emission filters. The percentage of nonviable cells was measured
using a modification of the method described by Rudolph et
al. (26). Base-line fluorescence F0 was
measured immediately after treatment with the corresponding medium and
addition of PI (30 µM). Subsequent fluorescence readings
were obtained at different times after the beginning of treatment. At
the end of the experiment, cells were permeabilized with 375 µM digitonin for 10 min at 37 °C to obtain the maximum
fluorescence corresponding to 100% of cell death
(Fmax). Percentage of cell death was calculated as follows: % cell death = 100 × (Fn Gene Expression Analysis--
Gene expression was assessed with
the differential display technique as described by Liang and Pardee
(21) using the RNA image kit from GenHunter. Total RNA from cerebellar
granule cells exposed to S Reverse Northern Dot Blot and Northern Blot--
To verify
overexpression of the cDNA fragments identified by differential
display, we used two different procedures: reverse Northern dot blot
and Northern blot. Bands excised from the gel may contain different
cDNA species. To identify the cDNA that is overexpressed in the
band, we used the reverse Northern dot blot technique (27). This
procedure allows the isolation and identification of cDNAs that
actually correspond to differentially expressed mRNAs, from bands
excised from a differential display gel. For reverse Northern dot blot
experiments, tetracycline-resistant colonies were randomly picked from
each plate and lysed by boiling in 50 µl of lysis buffer (0.1% Tween
20 in TE buffer, pH 8.0). The cloned cDNA fragments were amplified
using primers flanking the cloning site of the vector. Each PCR product
was dot-blotted onto duplicate nylon membranes using a microfiltration
system. The membranes were UV-cross-linked and probed with total
[32P]cDNA. The [32P]cDNA probes
were prepared by reverse transcription of 20 µg of total RNA obtained
from cerebellar granule cells control (S
Once cDNA overexpression was confirmed by reverse Northern dot
blot, we performed Northern blots to verify whether the selected cDNAs represent overexpression of a single mRNA. Northern blot experiments were also performed to study time course of NP1 expression. Total RNA was isolated using TRIzol reagent (Life Technologies, Inc.).
Denatured RNA samples (20 µg of total RNA) from cerebellar granule
cells controls (Sc+K+ and
S Preparation of NP1 and Data Base Searches and Nucleotide Alignment--
DNA sequencing
was performed with Thermo-Sequenase (Amersham Pharmacia Biotech) using
an ABI Prism 377 fluorescent sequencing instrument at the Serveis
Científico Tècnics (University of Barcelona, Barcelona,
Spain). Data base searches and sequence comparisons were performed
using BLAST and BLASTX search servers at the National Center for
Biotechnology Information (National Institutes of Health, Bethesda, MD).
SDS-PAGE and Western Blot Analyses--
After the corresponding
treatments, cerebellar granule cells were solubilized in lysis buffer
(5 mM Tris-HCl, 150 mM NaCl, 1% Igepal CA-630,
0.5% sodium deoxycholate, 0.1% SDS, 2 µg/ml aprotinin, 1 µg/ml
leupeptin, 100 µg/ml phenylmethylsulfonyl fluoride) and briefly
sonicated. The homogenate was centrifuged at 15,000 × g for 20 min at 4 °C. Total protein concentration was
determined using the BCA protein assay kit (Pierce). The polypeptides
were separated on 10% SDS-polyacrylamide gel electrophoresis and then electroblotted onto PVDF membranes (Millipore, Bedford, MA) according to the manufacturer's protocol. Blots were preincubated with 5% nonfat dry milk in Tris-buffered saline before immunostaining. For
specific immunodetection of NP1 protein, mouse anti-rat NP1 monoclonal
antibody (Transduction Laboratories, Los Angeles, CA) and rabbit
anti-rat polyclonal NP1 antibody (provided by Drs. Carsten Hopf and
Paul Worley, Department of Neuroscience, Johns Hopkins School of
Medicine, Baltimore, MD), were used at their appropriate concentrations
in a solution containing 0.5% nonfat dry milk and 1% bovine serum
albumin in Tris-buffered saline containing 0.1% Tween 20. Peroxidase-conjugated goat anti-mouse IgG (Transduction Laboratories,
Los Angeles, CA) or peroxidase-conjugated mouse anti-rabbit IgG (Sigma)
were used as secondary antibodies. In addition to the measurement of
the amount of protein before loading, we used a rabbit anti-actin
antibody (Sigma) to control for the amount of protein loaded.
Immunoreactive proteins were visualized using an enhanced
chemiluminescence detection system (ECL, Amersham Pharmacia Biotech).
Quantification of the intensity of the bands on the films was performed
with Kodak DS1 computer software. Densitometry values of the bands
representing NP1 immunoreactivity were normalized with the values of
the corresponding actin bands.
Statistical Analysis--
Results are expressed as mean ± S.E. of at least three separate experiments. Statistical significance
of differences was examined using independent t tests or
using one-way analysis of variance when required. Post
hoc multiple comparisons were performed using Student-Newman-Keuls tests.
Exposure to Low Potassium Induces Cell Death in Mature Cerebellar
Granule Cells--
Treatment of mature cerebellar granule cells with
low potassium resulted in a time-dependent increase in
neuronal death, measured with propidium iodide fluorescence (Fig.
1). The loss of neurons was ~60%,
24 h after switching from high to low extracellular concentration
of potassium. In comparison, in the same time period, control cultures
kept in conditioned medium containing serum and high potassium
(Sc+K+) exhibited 13% cell death
(Fig. 1). As additional controls, we used cultures in which conditioned
medium was replaced with fresh medium supplemented with high (25 mM) potassium, but without serum (S Neuronal Pentraxin 1 Gene Expression Is Up-regulated by Low
Potassium in Cerebellar Granule Cells--
To identify genes whose
expression is induced before neuronal death, we systematically compared
mRNA display patterns between cerebellar granule cells exposed to
high potassium (S
To further confirm that treatment of cerebellar granule cells with low
potassium induces overexpression of the NP1 gene, we performed Northern
blot analysis (Fig. 4A). The
cDNA fragment homologous to NP1 mRNA was
32P-labeled an used as hybridization probe. A single NP1
mRNA transcript was detected at ~5.5 kilobases, which is
consistent with the expected size of the NP1 mRNA (5341 bases)
(22). Comparison with untreated cultures
(Sc+K+) showed that overexpression
of the NP1 gene was induced only in cultures treated with low potassium
(S
To examine whether overexpression of NP1 mRNA is associated with an
increase in NP1 protein levels, we performed Western blot analyses
using a mouse monoclonal antibody against NP1. A unique band of
immunoreactivity of apparent molecular mass of ~50 kDa was detected
in extracts of cerebellar granule cell cultures, consistent with the
expected size of NP1 (22, 28). In cerebellar granule cells treated with
either conditioned medium or serum-free medium with high potassium, NP1
protein levels are low but detectable (Fig. 4C). However,
exposure to low potassium for 6 h induced a marked increase in the
levels of NP1 (Fig. 4C). Subsequent studies using a rabbit
polyclonal antibody against NP1 showed a unique band of
immunoreactivity and further confirmed that 6 h of treatment with
low potassium increases the levels of NP1 protein.
Time-course Analysis of NP1 mRNA Expression and Protein Levels
Induced by Low Potassium--
The coordinated program of biochemical
events that leads to cell death is activated immediately after exposure
to nondepolarizing culture conditions. However, cytoplasmic membrane
damage and neuronal loss is detected only after 8 h of exposure to
nondepolarizing culture conditions (Fig. 1). Thus, we studied the
expression of NP1 during the period of the first 8 h of exposure
to low potassium, before there is any significant membrane damage and
granule cell loss.
First, expression of NP1 mRNA was examined by Northern blot
analysis as a function of time of exposure to low potassium (Fig. 5A). Expression of NP1 gene in
cerebellar cultures was up-regulated at 2 h after potassium
deprivation and exhibited a peak of expression at 6 h of treatment
(Fig. 5B). The levels of
The levels of NP1 protein were also studied by Western blot analysis as
a function of time of exposure to low potassium (Fig. 5C). A
statistically significant increase in the levels of NP1 was first
observed after 4 h of potassium deprivation. The amount of NP1
protein peaked after 4 h of exposure to low potassium and the peak
levels were sustained for up to 8 h of treatment (Fig. 5D). The levels of actin protein did not significantly
change between treatments during the period investigated.
Antisense Oligodeoxyribonucleotides against NP1 Inhibit Low
Potassium Evoked Cell Death in Cerebellar Granule Cells--
To
examine the role of NP1 in the neurotoxicity induced by exposure to low
potassium, we used the antisense knock-down strategy. Treatment with an
antisense ODN (NP1AS) against NP1 mRNA significantly inhibited cell
death induced by 24 h of treatment with low potassium by ~60%
when compared with cultures treated with vehicle (Fig. 6A). In contrast, treatment
with the corresponding sense ODN (NP1S) was ineffective. At the same
time, treatment with antisense ODN against NP1, but not sense,
significantly reduced the levels of NP1 protein induced by potassium
deprivation by ~60%. Thus, the neuroprotective effect of the
antisense ODN against NP1 is preceded by inhibition of the
overexpression of NP1 evoked by low potassium (Fig. 6, B and
C).
Lithium Protects Mature Cerebellar Granule Cells from Cell Death
and Inhibits Overexpression of NP1 Protein Induced by Low
Potassium--
Acute treatment with lithium (5 mM)
significantly inhibited cell death induced by low potassium in mature
cerebellar granule cells. Lithium reduced cell death after 24 h of
treatment with low potassium by ~60%, when compared with the
respective control (Fig. 7A).
To determine whether the neuroprotective effects of lithium are
associated with modulation of NP1 expression, we performed Western blot
experiments in cerebellar granule cells treated with potassium
deprivation in the presence of lithium (Fig. 7B).
Densitometric analysis of the ratio between NP1 over actin
immunoreactivity showed that lithium significantly reduced by ~64%
the levels of NP1 protein evoked by low potassium treatment (Fig.
7C).
Consistent with results of previous studies (9, 13, 14), reduction
of the extracellular concentration of potassium induced a pronounced
cell death after 24 h of treatment (Fig. 1). Time-course analysis
of this process indicated that viability of cerebellar granule cells
was unaffected during the first 6 h of treatment. Significant cell
death was first detected only after 8 h of exposure to low
potassium, and this effect increased with time (Fig. 1). These results
are consistent with previous temporal analyses of the biochemical and
molecular events that occur during the process of cell death evoked by
low potassium in cerebellar granule neurons (9-11, 14, 29). Results
from these previous studies indicate that potassium deprivation induces a coordinated program of events before cerebellar granule cells commit
to die and apoptotic death becomes irreversible. Inhibitors of protein
and RNA synthesis reverse DNA fragmentation and block cerebellar
granule cell death when added within the first 3-4 h of treatment (14,
15). This indicates that "de novo" expression of death
genes, rather than inhibition of expression of survival genes, mediates
cell death by low potassium.
In the present study, we have investigated gene expression during the
early phase of the death program induced by potassium deprivation, to
identify genes specifically involved in neuronal death triggered by the
lowering of synaptic activity. Thus, after isolation and identification
of cDNAs differentially expressed by low potassium, we focused on
genes whose expression is restricted to the nervous system. First, the
analysis of differential gene expression revealed 102 bands that showed
a consistent difference between high and low potassium treatments.
Among these bands, 62 exhibited mRNA overexpression and only 12 bands attained the criteria of an at least 4-fold higher expression in
nondepolarizing than in depolarizing culture conditions. After
reamplification and subcloning, the cDNA species obtained from
these 12 bands were sequenced and compared with the GenBank data base.
Blast searches revealed that among the cDNAs with high homology to
known genes, only one of them (Fig. 3B), corresponding to
rat NP1 was expressed exclusively in the nervous system. Thus, in
subsequent experiments, we investigated the effect of treatment with
low potassium on NP1 expression.
Our results show that treatment of cerebellar granule cells with low
potassium induces a >15-fold increase of NP1 mRNA compared with
cells kept in high potassium either with or without serum (Fig.
5B). The increase in NP1 mRNA transcripts is followed by an increase in NP1 protein levels between 3- and 6-fold over control levels (Figs. 5D and 6C). This difference between
the amounts of NP1 message and protein is consistent with previous
findings showing that in cortical or cerebellar homogenates NP1 protein is rare (22). The discrepancy between the high amount of message and
the relatively low levels of NP1 protein has been interpreted as
indicating a rapid turnover, as might be expected for a secreted protein (22).
The increased expression of NP1 protein peaks after 4 h of
exposure to low potassium (Fig. 5, C and D). This
up-regulation of NP1 precedes cytoplasmic membrane damage by at least
4 h, and the peak of NP1 protein corresponds approximately to the
period when cerebellar granule cells commit to die (14). Induction of
other genes has been reported to occur before commitment to die. For
example, expression of c-Jun mRNA is induced rapidly and peaks at
2 h after survival signal withdrawal (15). Likewise, expression of
GAPDH reaches a maximum after 2 h of exposure to low potassium
(17). At present, the relationship between these genes and NP1 is
unknown, but comparison of their temporal pattern of expression after
treatment with low potassium suggests that NP1 is downstream of both
c-Jun and GAPDH expression.
To investigate whether overexpression of NP1 is directly involved in
low potassium-evoked cell death, we used the antisense knockdown
strategy. We found that antisense oligodeoxyribonucleotides against NP1
mRNA markedly inhibited the increase in NP1 protein and
significantly reduced cerebellar granule cell death evoked by low
potassium (Fig. 6). In contrast, NP1 sense oligonucleotides did not
significantly modify neither cell death nor NP1 protein levels induced
by low potassium (Fig. 6). These results strongly suggest that the
neuroprotective effects of these antisense oligonucleotides are due to
knockdown of NP1 gene expression and provide evidence to indicate that
NP1 is part of the cell death program induced by nondepolarizing
culture conditions in cerebellar granule cells.
Further evidence showing the involvement of NP1 in low potassium-evoked
cerebellar granule cell death was obtained after neuroprotective treatment with lithium. Treatments that inhibit low potassium-evoked apoptosis such as insulin-like growth factor or brain-derived neurotrophic factor suppress expression of cell death genes by activating different intracellular signaling pathways (30-32). Likewise, lithium promotes survival of mature cerebellar neurons (33)
by a dual mechanism comprising increased transcription of survival
genes such as bcl2 and inhibition of expression of pro-apoptotic genes such as bax and p53 (34-36).
In the experiments reported here, we found that acute treatment with
lithium significantly inhibits the expression of NP1 evoked by low
potassium and this effect is followed by a significant reduction of
cerebellar granule cell death (Fig. 7). Thus, inhibition of NP1
expression by lithium is associated with the neuroprotective effects of
this drug.
The pentraxin family of proteins may be divided into two structural
classes based on size (see Ref. 37 for review). Recent evidence
indicates that long pentraxins such as NP1, which share a high homology
at the C-terminal half, possess diverse functions. Thus, NP1 was
originally identified as a calcium-dependent binding protein for the snake venom toxin taipoxin and was found to be expressed exclusively in neurons (22). Based on NP1's homology to
short pentraxins such as C-reactive protein and serum amyloid P
protein, the function of NP1 has been proposed to mediate the uptake of
synaptic material during synapse remodeling (22, 28, 38). The results
reported here showing that NP1 is involved in cerebellar granule cell
death induced by nondepolarizing culture conditions provide evidence of
a new function for NP1. On the other hand, neuronal activity-related
pentraxin (Narp), also called neuronal pentraxin 2, another member of
the long pentraxin family, has been proposed to mediate the synaptic
clustering of In summary, the present results show that treatment with low potassium
induces overexpression of NP1 before cell death. In addition,
inhibition of NP1 overexpression by either antisense oligodeoxyribonucleotides or lithium prevents cerebellar granule cell
death evoked by low potassium. These findings provide evidence of a new
function for NP1 and indicate that NP1 is part of the gene program that
leads to apoptotic cell death in cerebellar granule cells kept under
nondepolarizing culture conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
K+) or
fresh unconditioned serum-free medium containing 5 mM
potassium (S
K
). Immediately after
replacement, cells were incubated at 37 °C for different times up to
24 h.
F0)/(Fmax
F0), where Fn is
fluorescence at any given time. Cells were kept in the incubator between measurements. In some experiments, percentage of dead cells was
measured counting PI stained over total number of cells using
simultaneous fluorescence and phase contrast observation in an
epifluorescence microscope. In these experiments, cells were incubated
with 10 µM PI for 30 min and fixed in 3.7%
paraformaldehyde for 20 min at room temperature before addition of a
final glycerol protective layer.
K+ and
S
K
for 2 and 4 h was isolated using
the Rneasy mini kit (Qiagen) and treated with DNase I (GenHunter,
Nashville, TN). First strand cDNA synthesis and
33P-radiolabeled differential display PCR were performed
using the RNA image kit (GenHunter). The PCR reactions were performed
in duplicate for each treatment. The amplified cDNAs were resolved by electrophoresis using a 6% denaturing polyacrylamide gel. After immobilizing the gel on Whatman no. 3MM paper and drying it for 30 min
under vacuum at 80 °C, the gel was exposed to X-Omat AR film
(Eastman Kodak Co.) overnight. The autoradiogram and the dried gel were
oriented with needle punches to be able to locate in the gel the bands
identified in the film. After developing the film, the patterns of
amplified cDNA bands were compared among treatments. We chose
cDNA bands exhibiting a higher intensity in
S
K
compared with
S
K+, which indicates that low potassium
treatment induces the overexpression of a gene represented in the band.
The cDNA bands of interest were excised from the gel, reamplified
by PCR with the same set of primers and PCR conditions used in the
mRNA display but with a higher concentration of dNTPs, and ligated
into the pCR-TRAP cloning vector (GenHunter). Ligated plasmids were
transformed into GH-competent cells and plated on LB plates containing
20 µg/ml tetracycline. The pCR-TRAP vector includes a
tetracycline-dependent positive selection of plasmids with
DNA inserts. Only recombinant plasmids confer antibiotic resistance.
K+)
and treated with low potassium (S
K
) for
4 h, in the presence of [
-32P]dCTP (3000 Ci/mmol,
PerkinElmer Life Sciences). Equal counts (5-10 × 106 cpm) of the cDNA probes from each treatment,
(S
K+) and (S
K
),
were heat-denatured and used to probe the duplicate blots.
K+) and treated with low potassium
(S
K
) were electrophoresed in 1.3% agarose
and 0.66 M formaldehyde gels, transferred to nylon
membranes (Hybond-XL, Amersham Pharmacia Biotech), and the RNA was
fixed to the membranes by baking for 2 h at 80 °C.
Hybridization with 32P-labeled probes and washing
conditions were performed as described by the membrane manufacturer.
Filters were exposed to BioMax films (Amersham Pharmacia Biotech) with
intensifying screens for 12-48 h at
80 °C.
-Actin Probes--
The NP1 probe used
for Northern blot analysis corresponds to nucleotides 5127-5339 of the
NP1 cDNA. The NP1 probe was obtained by PCR amplification of a
positive clone identified by reverse Northern dot blot. The PCR product
was electrophoresed in agarose and purified using the QIAEX II gel
extraction kit (Qiagen) and 32P-labeled using Ready-To-Go
DNA labeling beads (Amersham Pharmacia Biotech). The
-actin probe
was obtained by digestion of a pUC19 vector containing a 1.9-kilobase
pair human
-actin insert between BamHI sites.
Densitometric values of NP1 mRNA bands were obtained using Kodak
DS1 computer software and were normalized with the densitometric values
of the corresponding
-actin mRNA band.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
K+). In these cultures, cell death was 20%
after 24 h of treatment. Serum removal did not have a significant
effect on cell death until after 12 h of treatment. Moreover,
after 24 h of treatment, cell death by serum removal was only 7%
compared with undisturbed Sc+K+
controls. Treatment with low potassium (5 mM) did not
produce any significant cell death until after 8 h of treatment
compared with controls. However, 24 h of exposure to low potassium
(S
K
) induced 40% cell death, compared with
exposure to serum-free medium containing high potassium
(S
K+). To identify genes of the death program
activated by low potassium before there is significant cell death, we
investigated differential gene expression in cells treated with
S
K+ and S
K
for 2 and 4 h. We chose these time points because they are well before
any significant differences in cell death can be detected between these
two treatments (Fig. 1). Moreover, previous studies have shown that
cerebellar granule cells can be rescued from low potassium-induced cell
death if RNA synthesis inhibitors are added within the first 4 h
of treatment but not after (14, 15).
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Fig. 1.
Time course of neuronal death by low
potassium in mature cerebellar granule cells. Cerebellar granule
neurons were cultured in basal Eagle's medium supplemented with 25 mM KCl and 10% fetal bovine serum for 8 days. Conditioned
medium (Sc+K+), was replaced with
one of the following media: fresh unconditioned serum-free medium
supplemented with 25 mM potassium
(S K+) or fresh unconditioned serum-free
medium containing 5 mM potassium
(S
K
). Immediately after medium replacement,
cells were incubated at 37 °C for different times up to 24 h.
Time course of cell death was assessed by PI fluorescence at the
indicated times after initiation of treatment and expressed as a
percentage of maximum cell death obtained with digitonin. Values are
mean ± S.E. of three independent experiments. *, significantly
different from control cultures
(Sc+K+). p < 0.05, using one-way analysis of variance followed by Student-Newman-Keuls
test for each time.
K+), as controls, and cells
treated with low potassium (S
K
). RNA from
cultures treated with either S
K+ or
S
K
was isolated after 2 and 4 h of
treatment and subjected to differential gene display analysis.
33P-labeled PCR was performed with 24 different 5'
arbitrary primers combined with each one of three different 3'-anchored
primers, for a total of 72 different primer combinations. PCR reactions were performed in duplicate for each treatment group and time point.
The analysis of differential gene expression was performed by comparing
the band pattern of all these primer combinations for each treatment.
We chose only those bands that showed a consistent differential
expression, in both duplicates and in the two time points analyzed,
between the two treatments. We found 102 bands that in both duplicates
exhibited consistent differential expression between high and low
potassium treatments at both 2 and 4 h of treatment. Among all of
these, 62 bands indicated mRNA overexpression. We chose only those
bands that exhibited at least a 4-fold higher densitometric intensity
in low over high potassium treatments. We reamplified, subcloned, and
sequenced the cDNA species from 12 bands that attained the
criterion difference between high and low potassium treatments at both
time periods. Finally, we isolated a cDNA band that showed
overexpression after both 2 and 4 h of low potassium treatment,
named AP21G1 (Fig. 2). To identify the cDNA fragment overexpressed in this band, several clones obtained from the reamplified cDNA from this band were subjected to reverse Northern dot blot (Fig. 3A).
Reverse Northern analysis showed that 4 out of 5 clones (AP21G1-1, -2, -3, and -5) obtained from the AP21G1 band exhibited higher signal when
hybridized with cDNA from low potassium treated cultures than when
hybridized with cDNA from control cultures (Fig. 3A).
This result indicates that these clones correspond to the gene whose
overexpression was induced by treatment with low potassium and,
therefore, the gene that confers the high intensity signal to the
AP21G1 band (Fig. 2). PCR amplification of the inserts of the positive
clones revealed a fragment of ~200 base pairs in the four cDNAs.
The sequence of the cDNA inserts of the four positive clones was
identical, confirming that reverse Northern actually identified a
single cDNA that was overexpressed in the AP21G1 band. The sequence
of this cDNA overexpressed by low potassium treatment is 213 base pairs long and it is represented in Fig. 3B. A search of the
GenBankTM data base at the National Center for Biotechnology
Information using the BLAST program revealed that the AP21G1 fragment
is 100% homologous with the 3'-untranslated region of rat NP1 mRNA
(GenBankTM accession no. U18772) (22).
View larger version (133K):
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Fig. 2.
Differential display analysis of gene
expression between high and low extracellular potassium treatment in
cerebellar granule cells. Total RNA was extracted from 8DIV
cerebellar granule cultures after 2 and 4 h of high
(S K+) or low potassium
(S
K
) treatments and subjected to
differential display analysis. The autoradiogram shows the band pattern
obtained from [
-33P]dATP-labeled differential display
reactions performed in duplicate using 5'-AAGCTTTTTTTTTTTG-3' as an
anchored primer (H-T11G) and 5'-AAGCTTTCTCTGG-3'as a random
arbitrary primer (H-AP21). The arrow indicates a band
(AP21G1) corresponding to a cDNA fragment that is overexpressed
both after 2 h (lanes 3 and 4)
and 4 h (lanes 7 and 8) of
potassium deprivation.
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Fig. 3.
Reverse Northern dot blot of clones from
AP21G1 band. A, the cDNA from the band AP21G1
represented in Fig. 2 (lane 8) was excised,
reamplified, and ligated into the pCR-TRAP cloning vector. The PCR
products from five randomly picked colonies (AP21G1-1, -2, -3, -4, and
-5) were blotted onto duplicate filters. One of the filters
(S K+) was hybridized with
32P-labeled cDNA from cerebellar granule cell cultures
control. The other filter was hybridized with 32P-labeled
cDNA from cerebellar granule cells treated with low potassium for
4 h (S
K
). B, nucleotide
sequence of AP21G1-1 clone cDNA fragment. The cDNA inserts of
the AP21G1-1, -2, -3, and -5 clones showing differential expression
were sequenced. The nucleotide sequence of the four cDNA inserts
was identical. The sequence of the primers used in differential gene
display analysis are underlined (H = HindIII site at the 5' end of the primers).
K
). The level of NP1 expression in
cultures subjected to serum deprivation (S
K+)
was as low as in untreated cells. Densitometric analysis of Northern
blot autoradiograms indicated that the ratio of NP1 over
-actin
mRNA was ~10-fold higher in cultures exposed to low potassium for
4 h compared to both Sc+K+ and
S
K+ controls (Fig. 4B).
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Fig. 4.
Low potassium increases NP1 protein and
mRNA levels in mature cerebellar granule cell cultures.
A, Northern blot analysis showing overexpression of NP1
mRNA after 4 h of potassium deprivation. Conditioned medium
(Sc+K+), was replaced with one of
the following media: fresh unconditioned serum-free medium supplemented
with 25 mM potassium (S K+) or
fresh unconditioned serum-free medium containing 5 mM
potassium (S
K
). Total RNA (20 µg/lane)
was isolated for each treatment and run in a 1.3% agarose gel and
transferred to nylon filters. The filters were hybridized with the
32P-labeled AP21G1-1 cDNA fragment homologous to NP1
identified by differential display. The blot was also rehybridized to
-actin probe as control. B, quantitative analysis of NP1
mRNA expression normalized with
-actin mRNA. The
autoradiographic signal intensities were determined by densitometric
analysis. The ratio of NP1 over
-actin intensity was expressed as
percentage of control values. Values are the mean ± S.E. of three
independent experiments. *, significantly different from control values
(Sc+K+). p < 0.05, Student's t test. C, Western blot showing the
immunoreactivity of two different antibodies against NP1. Proteins were
extracted from undisturbed mature cerebellar granule cells
(Sc+K+), or after 6 h of high
(S
K+) or low potassium
(S
K
). Protein extracts were run in a 10%
SDS-PAGE and transferred to PVDF membranes. Membranes were incubated
with two different primary antibodies: a mouse anti-NP1 monoclonal
antibody and a rabbit anti-NP1 polyclonal antibody. Chemiluminescent
detection shows a unique band of ~50 kDa. Actin was used as control
for protein loading.
-actin mRNA remained relatively constant throughout the time course examined.
View larger version (34K):
[in a new window]
Fig. 5.
Time-course analysis of NP1 mRNA
expression and protein levels. A, Northern blot showing
NP1 mRNA expression before any treatment
(Sc+K+) and after 2, 4, 6, and
8 h of high (S K+) and low potassium
(S
K
) treatments. Total RNA (20 µg/lane)
from each treatment and each time point, as indicated at the top of the
autoradiograms, was run in a 1.3% agarose gel and transferred to nylon
membranes. 32P-Labeled NP1 and
-actin were used as
hybridization probes in the same membranes. This is a representative
Northern blot out of three independent experiments. B,
quantitative analysis of the time course of NP1 mRNA expression
normalized with
-actin mRNA. The autoradiographic signal
intensities were determined by densitometric analysis. The ratio of NP1
over
-actin intensity was expressed as percentage of control values.
The analysis was performed in three independent experiments with
qualitatively similar results. C, Western blot showing the
levels of NP1 protein before any treatment
(Sc+K+) and after 2, 4, 6, and
8 h of high (S
K+) or low potassium
(S
K
) treatments. Protein extracts were run
in a 10% SDS-PAGE and transferred to PVDF membranes. Membranes were
incubated with mouse anti-NP1 antibody and chemiluminescent detection
shows a unique band of ~50 kDa. Actin was used as control for protein
loading. D, quantitative analysis of NP1 protein normalized
using actin levels. The autoradiographic signal intensities were
determined by densitometric analysis. The ratio of NP1 over actin
intensity was expressed as percentage of control values. Values are the
mean ± S.E. of three independent experiments. *, significantly
different from control values
(Sc+K+). p < 0.05, Student's t test.
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Fig. 6.
NP1 antisense ODNs inhibit cerebellar granule
cell death evoked by low potassium. Mature (8DIV) cerebellar cells
were incubated in serum-free medium supplemented with 25 mM
potassium (S K+), or serum-free medium
containing 5 mM potassium (S
K+).
Antisense ODNs (NP1AS, 10 µM, AS) or
corresponding sense ODNs (NP1S, 10 µM, S) were
added immediately after switching from high to low extracellular
concentration of potassium. A, neuroprotective effect of NP1
antisense ODNs on neuronal death by low potassium. The number of dead
cells was assessed by PI staining after 24 h of exposure to the
different treatments. Values are the mean ± S.E. of three
independent experiments. +, significantly different from
S
K
; *, significantly different from
S
K+. p < 0.05, using
independent t test analysis. B, Western blot
showing the effect of antisense ODNs on the increase of NP1 protein
levels evoked by low potassium. Protein extracts were run in a 10%
SDS-PAGE and transferred to PVDF membranes. Membranes were incubated
with mouse anti-NP1 antibody. Actin was used as control for protein
loading. C, quantitative analysis of the effects of
antisense and sense ODNs on the increase of NP1 protein levels evoked
by low potassium. NP1 protein was normalized with actin. The
autoradiographic signal intensities were determined by densitometric
analysis. The ratio of NP1 over actin intensity was expressed as
percentage of control values. +, significantly different from
S
K
; *, significantly different from
S
K+. p < 0.05, using
independent t test analysis
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Fig. 7.
Effects of lithium on cell death and NP1
protein levels evoked by low potassium in mature cerebellar granule
neurons. Mature (8DIV) cerebellar cells were incubated in high
(S K+), or low potassium
(S
K+). Lithium (Li, 5 mM) was added in both treatments as indicated.
A, lithium inhibits cell death evoked by low potassium. Cell
death was assessed with PI fluorescence after 24 h of treatment.
Cell death was expressed as a percentage of maximum cell death obtained
with digitonin. Values are mean ± S.E. of three independent
experiments. *, significantly different from
S
K+; +, significantly different from
S
K
. p < 0.05, Student's
t test. B, Western blot showing the effects of
lithium on the increase of NP1 levels evoked by low potassium. Protein
extracts were run in a 10% SDS-PAGE and transferred to PVDF membranes.
Membranes were incubated with mouse anti-NP1 antibody. Actin was used
as control for protein loading. C, quantitative analysis of
the effects of lithium on the increase of NP1 protein levels evoked by
low potassium. NP1 protein was normalized with actin. The
autoradiographic signal intensities were determined by densitometric
analysis. The ratio of NP1 over actin intensity was expressed as
percentage of control values. +, significantly different from
S
K
; *, significantly different from
S
K+. p < 0.05, using
independent t test analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino-3-hydroxy-5-methyl-4-isoxazole propionate
glutamate receptors at a subset of excitatory synapses, supporting
thereby a synaptogenic signaling function (39, 40). Interestingly, Narp
is rapidly induced and dynamically regulated by depolarizing conditions
(39, 40). However, in contrast with the marked increase that treatment
with low potassium evokes on NP1 expression, such treatment did not
have the same effect on the expression of Narp (results not shown).
This raises the possibility that Narp and NP1 could function as part of
genetic switch to sensor changes in synaptic activity; Narp associated with neurite outgrowth after depolarization and NP1 associated with
neuronal death under nondepolarizing conditions. Experiments using NP1
and Narp gene transfection into host neurons will allow to investigate
this hypothesis and to determine whether NP1 overexpression is
sufficient to elicit its apoptotic effect or if it requires the
concerted action of other cellular proteins.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Carsten Hopf and Dr. Paul Worley for providing the rabbit antibody against NP1. We thank Juana María Hurtán for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by Comisión Interministerial de Ciencia y Tecnología Grant SAF98-0063, Plan Nacional I+D, Ministerio de Educación y Cultura of Spain (to R. T.) and by a concerted research contract from "Centro para el Desarrollo Tecnológico Industrial," Ministerio de Industria-Merck Farma y Química of Spain.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.
Supported by a "Formación de Personal Investigador"
fellowship from "Subdirección General de Formación
y Promoción del Conocimiento, Ministerio de Educación
y Cultura" of Spain.
§ Supported by the "Programa de Reincorporación" of the "Secretaría de Universidades e Investigación" of Spain.
¶ To whom correspondence should be addressed. Tel.: 3493-3638303; Fax: 3493-3638324; E-mail: rtonbi@iibb.csic.es.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M007967200
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ABBREVIATIONS |
---|
The abbreviations used are: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NP1, neuronal pentraxin 1; ODN, oligodeoxyribonucleotide; PI, propidium iodide; Narp, neuronal activity-related pentraxin; DIV, days in vitro; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
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