From the
Postmitotic sympathetic neurons are known to undergo a
programmed cell death (apoptosis) when they are deprived of nerve
growth factor (NGF) or treated with arabinofuranosyl nucleoside
antimetabolites. Here we report the existence of a biochemical
mechanism for the induction of neuronal death by an endogenous
nucleoside in the presence of NGF. In support of such a mechanism we
show that 2-deoxyadenosine (dAdo) induces apoptosis in chick embryonic
sympathetic neurons supported in culture by NGF, excess
K 2`-Deoxyadenosine (dAdo)( Clinical reports indicate that SCIDS also affects neurological
function in children(12, 13) . Furthermore,
life-threatening disorders of the central nervous system, as well as
hepatic, renal, and respiratory functions, have been encountered in
patients receiving ADA inhibitors as immunosuppressants(14) .
However, unlike the extensive studies carried out on cultured lymphoid
cells to improve understanding of immune function in ADA-deficient
patients, there are no parallel reports on the effects of dAdo on
neuronal systems. Primary culture of post-mitotic sympathetic neurons
derived from paravertebral ganglia of chick embryo has proved to be a
useful model to study a variety of neuronal properties including the
toxicities of
NGF-supported cultures of sympathetic neurons showed
inhibitory effects of dAdo on neurite extension as early as 6-8 h (Fig. 1). Photomicrographs in Fig. 2demonstrate that 300
µM dAdo completely arrested neurite outgrowth from almost
all the neurons within the first 24 h when they were maintained in the
presence of NGF (Fig. 2B). In the next 24 h, the cell
bodies of the neurons disintegrated in the presence of dAdo (Fig. 2D) whereas the untreated neurons showed the
normal, extensive neuritic growth (Fig. 2, A and C). Inhibition of neurite growth and subsequent death was
concentration-dependent and maximal at 300 µM (Fig. 3A). Related compounds such as
2-deoxyguanosine, guanosine, inosine, and hypoxanthine at
concentrations up to 500 µM did not affect neuronal growth
and survival (not shown).
Figure 1:
2`-Deoxyadenosine arrests neurite
growth in sympathetic neurons. Photomicrographs of sympathetic neurons
grown for 8 h in control medium (A) show almost all cells
extending neurites. In the presence of 300 µM dAdo (B), only bright, light refractory cell bodies are visible.
The scalebar represents 50
µm.
Figure 2:
2`-deoxyadenosine-induced apoptosis in
sympathetic neurons. Photomicrographs A-D are of
sympathetic neurons grown for 24 h (A and B) or 48 h (C and D) in control medium (A and C) or in the presence of 300 µM dAdo (B and D). Photomicrographs E-I are of
neurons treated with fluorescent markers for condensation and
fragmentation of DNA using bisbenzimide (E-G) and the
TUNEL method (H and I) after 2 days in culture in the
absence (E and H) or presence (F, G, and I) of 300 µM dAdo. Arrows indicate fluorescence typical of apoptotic cells. The
photomicrographs are representative of 3-15 observations under
each condition. The scalebars represent 50
µm.
Figure 3:
Inhibition of adenosine deaminase and
nucleoside transporter do not affect 2`-deoxyadenosine actions. A, neuronal loss in cultures containing increasing
concentrations of dAdo alone (solidline) or with 3
µM deoxycoformycin (brokenline)
expressed as a percentage of neurons in matched untreated control
dishes. B, uptake and retention of
[
Because programmed cell death (apoptosis)
plays an important role in controlling neuronal populations in
vivo(20) , and dAdo is an endogenous product of DNA
turnover and may reach high levels under pathological conditions (2, 3) we questioned whether dAdo could induce
apoptosis in neurons. Apoptosis was identified using two methods. The
nuclear dye bisbenzimide was used to detect condensation of nuclear
chromatin(21) . Bisbenzimide produced faint and diffuse
fluorescence from untreated control neurons (Fig. 2E),
but after dAdo treatment the fluorescence was more intense and
punctate, indicating condensation of the chromatin material (Fig. 2, F and G). The other method used is
specific for detecting neucleosomal breaks in DNA where 3-OH ends of
DNA fragments bind to digoxigenin-conjugated nucleotides to produce a
characteristic yellow-green fluorescence. Several of the remaining
cells after dAdo treatment show the characteristic apoptotic type
fluorescence (Fig. 2I), whereas almost all neurons
grown in control medium were negative for fragmented DNA (Fig. 2H). Cells showing apoptotic type fluorescence
were counted in control and dAdo-treated cultures. Neurons positive for
apoptosis represented less than 1% of the population in control
cultures compared to 31% in dAdo-treated cultures. Although NGF
deprivation is known to produce apoptotic death of sympathetic neurons,
dAdo-mediated toxicity was not a consequence of blockade of the actions
of NGF at the receptor level or along its intracellular signaling
pathway because neurons supported in culture by a diverse group of
neurotrophic agents were also sensitive to the lethal effects of dAdo (Table 1).
Compared to adenosine, dAdo is virtually inert in
terms of its ability to activate membrane nucleoside receptors to
influence the intracellular cAMP signaling system. Therefore, we
considered the possibility of an intracellular site of action in
apoptosis. When freshly plated neurons were incubated with
[ Consistent with the uptake data, inhibition of the nucleoside
transporter (10 µM NBTI) did not protect neurons against
dAdo-induced toxicity (Table 2). Toxic effects of dAdo in human
lymphocytes and other cell types have been related to blockade of DNA
synthesis or NAD synthesis, because dAdo toxicity is reversed by excess
uridine or 2`-deoxycytidine or by excess nicotinamide to maintain NAD
levels(22, 23) . However, 100 µM 2`-deoxycytidine (Fig. 4A) or 3 mM
nicotinamide (Fig. 4B) did not prevent the lethal
action of dAdo on neurons. Higher concentrations of 2`-deoxycytidine or
nicotinamide or up to 300 µM uridine also failed to
protect neurons exposed to 300 µM dAdo (Table 2).
Figure 4:
Importance of nucleoside kinase in
2`-deoxyadenosine toxicity. Photomicrographs of neurons grown for 2
days in the presence of 300 µM dAdo plus 100 µM 2-deoxycytidine (A), 3 mM nicotinamide (B), 0.1 nM ITu (C), or 3 nM ITu (D), representative of 3-5 observations under each
condition.
Deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl) adenine are potent
inhibitors of ADA (24) used to mimic immunodeficiency syndrome
in experimental models(11, 25) . However, neither
erythro-9-(2-hydroxy-3-nonyl) adenine nor deoxycoformycin modified the
toxic effects of dAdo in our neuronal model (Table 3; see also Fig. 3A). These results are significantly different
from those reported for human lymphocytes and several other cell lines
commonly used in cancer research, and raise an important question about
the metabolic disposition of dAdo in sympathetic neurons as compared to
other types of cells (see below).
Since inhibition of adenosine
deaminase did not facilitate the toxic effects of dAdo and since agents
that protect against dAdo toxicity in other cells were without effect,
we considered the possibility that nucleoside kinases might play a
major role in the metabolism of dAdo in sympathetic neurons.
5`-Iodotubercidin (ITu), an inhibitor of adenosine kinase(26) ,
was used to test this idea. Remarkably, as little as 0.1 nM ITu offered some protection (Fig. 4C), and 3
nM ITu almost completely blocked the lethal effects of 300
µM dAdo (Fig. 4D). This effect was not due
to blockade of dAdo transport, because as high as 3 µM ITu
had no effect on [ The above experiments strongly
suggest that the most efficient metabolic pathway for dAdo may be
phosphorylation to dATP which could then be responsible for
dAdo-induced arrest of neurite growth, DNA fragmentation, and cell
death. In support of such a mechanism, we found a more than 40-fold
increase in dATP content of neurons within 2 h of exposure to 300
µM dAdo (34.96 ± 5.98 versus 0.75 ±
0.16 pmol of dATP/µg of protein in treated and control cultures,
respectively, n = 9). The formation of dATP from dAdo
was both time-dependent (Fig. 5A) and
concentration-dependent (Fig. 5B) with maximum dATP
levels achieved following incubation with 100 µM dAdo. The
elevation of intracellular dATP was dose-dependently and almost
completely blocked (83 ± 1%) by 3 nM ITu (Fig. 5C), consistent with the dose-dependent
protective action of ITu against dAdo toxicity. These data support the
idea that sympathetic neurons rapidly phosphorylate intracellularly
accumulated dAdo to its toxic product.
Figure 5:
Expansion of dATP pool following dAdo
exposure. Panels A-C show the 2`-deoxy ATP (dATP)
content of neurons exposed to dAdo. Neurons grown for 2-3 days
were exposed to 300 µM dAdo for increasing time periods (A) or to various concentrations of dAdo for 2 h (B)
to determine dATP content in treated and matched control (none) dishes. PanelC shows dATP content of
neurons exposed to 300 µM dAdo in the absence (0)
and presence of increasing concentrations of the adenosine kinase
inhibitor ITu compared to untreated control (cont) dishes.
Values represent the mean (± S.E.) of 3-7 experiments
under each condition. ITu concentrations from 0.3 to 30 nM each caused a significant decrease in dATP formation (p < 0.01 compared to 0 ITu, Student's t-test).
The lethal effects of dAdo were extensively studied over the
past 20 years exclusively in blood cells, and our report shows that
neuronal cells are equally susceptible to this endogenous nucleoside.
This dramatic parallel is as impressive as the differences in the way
the nucleoside is metabolized by the two types of cells. The most
noticeable differences were the absence of a specific carrier or
transport mechanism for the nucleoside in the plasma membrane, a minor
role of adenosine deaminase, and the inability of nicotinamide and
pyrimidines to reverse the dAdo toxicity in neurons. Whether neuronal
cells have adopted these diversities to safeguard against the toxicity
of the nucleoside or have acquired a unique nucleoside metabolic
pathway for maintaining their populations is an intriguing question. Sympathetic neurons differ from non-neuronal cells, which are
protected against dAdo toxicity by inhibitors of nucleoside transport (27) and by mutations resulting in lack of transporter
expression(28) . Consistent with the lack of protection against
dAdo toxicity by nucleoside transport inhibitors, the uptake of
[ Regardless of the exact mechanism of dAdo uptake, we have
demonstrated rapid accumulation of very high levels of dATP within
1-8 h of exposure to dAdo. This was associated with complete
arrest of neurite growth even though neuronal soma appeared viable.
Neuronal death and evidence of apoptosis were prominent after
24-48 h. These findings are consistent with those showing that
neurite degeneration and somatic apoptosis are independent events in
sympathetic neurons deprived of NGF (34) and suggest that dAdo
may exert its toxicity by more than one mechanism. Data showing that
inhibition of nucleoside kinases by nanomolar concentrations of Itu not
only blocked dATP formation but also completely protected neurons
against toxicity of 300 µM dAdo, offer strong evidence
relating dATP formation to neuronal toxicity and death. However, there
was a discrepancy between the concentration of dAdo needed to produce
maximum elevation of dATP ( Disturbance of
nucleoside metabolism by arabinofuranosyl nucleoside antimetabolites (e.g. cytosine arabinoside) has been shown to produce
apoptosis in sympathetic neurons(35, 36) , and
analogues of cyclic nucleotides rescue sympathetic neurons from
apoptosis following NGF withdrawal(21, 37) . The
present findings show for the first time that an endogenous nucleoside
is also capable of inducing apoptotic death in sympathetic neurons in
the presence of NGF or other diverse factors capable of supporting
survival of sympathetic neurons in culture(38, 39) .
The extraordinary protection afforded by nanomolar concentrations of
ITu against submillimolar concentrations of dAdo and the lack of effect
of ADA inhibitors indicate that the primary fate of dAdo in sympathetic
neurons is phosphorylation rather than deamination. Whether nucleoside
kinase or deaminase activities change during development to serve as a
positive or negative signal for neuronal survival remains to be
established. In any event, our study draws attention to the importance
of purine and pyrimidine metabolism in a neuronal system in maintaining
growth and survival. Understanding of these metabolic pathways may
provide important insights to mechanisms involved in modeling the
nervous system during development as well as in neurodegenerative
disease. Johnson and colleagues (21, 40) and others (41) have obtained extensive evidence to establish that
sympathetic neurons undergo a programmed cell death that requires
protein synthesis as well as mRNA synthesis and is accompanied by
nuclear DNA fragmentation. While the majority of these studies are
related to the inhibition of programmed cell death by NGF, it has been
suggested that cytokines may regulate sympathetic neurons by initiating
programmed death(42) . We have presented evidence that a
deoxynucleoside is also able to initiate apoptosis in sympathetic
neurons in the presence of NGF. Thus, neurons appear to have two or
more apoptotic mechanisms to regulate their population, either limited
supply of a protective factor (NGF) or an excess of initiating
nucleosides. It is tempting to speculate that one or both of these
pathways could be active during development or pathologic
neurodegeneration.
, phorbol 12,13-dibutyrate, or forskolin. Neuronal
death was related to a dramatic increase in the dATP content of
sympathetic neurons exposed to dAdo (34.96 ± 5.98 versus 0.75 ± 0.16 pmol/µg protein in untreated controls, n = 9), implicating dATP in the toxicity. Supportive
evidence for a central role of dATP was gained by inhibition of kinases
necessary for phosphorylation of dAdo. 5`-Iodotubercidin in nanomolar
concentrations completely and dose-dependently inhibited formation of
dATP and also protected against toxicity of submillimolar
concentrations of dAdo in sympathetic neurons. Although some of these
actions of dAdo were remarkably similar to those reported for human
lymphoid cells, several were uniquely different. For example,
[
H]dAdo was not transported into neurons by the
nucleoside transporter, and therefore inhibition of the transporter
(dilazep, nitrobenzylthioinosine) did not prevent neurotoxicity by
dAdo. Precursors of pyrimidine synthesis (2`-deoxycytidine, uridine) or
NAD
synthesis (nicotinamide) were ineffective in
protecting sympathetic neurons against dAdo toxicity. Finally,
inhibition of adenosine deaminase by deoxycoformycin or
erythro-9-(2-hydroxy-3-nonyl) adenine did not potentiate the toxic
effects of dAdo. Our results provide evidence for the first time that
neuronal cells are as susceptible to nucleoside lethality as human
lymphocytes are, and provide a new model to study the salvage pathway
of deoxyribonucleosides in controlling neuronal populations through
programmed cell death.
)
has received
major attention because of its central role in severe combined
immunodeficiency syndrome (SCIDS). The nucleoside is highly toxic to
human lymphocytes and is responsible for dysfunction of the immune
system(1, 2, 3, 4) . Normally, dAdo
derived from degraded DNA and dietary sources is rapidly metabolized by
adenosine deaminase (ADA) in lymphoid and other tissues. However, in
the absence of ADA, as in a genetic disorder responsible for SCIDS, the
concentration of dAdo increases in blood and tissues (5, 6) to induce a wide range of toxicities. Numerous
studies have employed in vitro models to examine the mechanism
of lethal actions of nucleosides in lymphocytes, lymphoblasts,
fibroblasts, HeLa cells, and other cell
types(7, 8, 9, 10) . One of the
consistent observations was the enhancement of dAdo toxicity after
treatment of cells with the ADA inhibitor deoxycoformycin, establishing
a link between clinical and biochemical findings(11) .
-amyloid and
1-methyl-4-phenylpyridinium(15, 16) . Therefore, we
selected this model to evaluate the effects of dAdo and other agents on
neuronal survival and growth in the presence of nerve growth factor
(NGF) and other trophic agents.
Cell Culture
Sympathetic neurons were obtained
from paravertebral, lumbosacral ganglia of 11-day-old chick embryos and
prepared for cultures as described(17) . Neurons were plated in
35-mm plastic culture dishes or glass coverslips for fluorescence
staining coated with polylysine (100 µg/ml for 3-4 h)
containing 1 ml of Dulbecco's modified Eagle's medium plus
F-12 (1:1) supplemented with 5 µg/ml insulin and transferrin, 0.5%
chick embryo extract, and 50 ng/ml NGF. In some experiments, NGF was
replaced by other neurotrophic factors, as described under
``Results.'' About 10,000 neurons were plated for
morphological studies. The cell number was increased to about
80,000/plastic dish for biochemical assays. The number of surviving
neurons was quantified by scanning along a strip with an area of 1/24th
of the total surface of the culture dish using a Nikon Diaphot
phase-contrast microscope (100). Generally, neurons with
refractory cell bodies and neurites of at least 3-4 times the
length of cell bodies were considered as live cells. However, several
treatments arrested the neurite growth without killing the cell bodies
(trypan blue-negative) within the first 18-24 h, and caused cell
body disintegration (ethidium bromide-positive) in the next 24-48
h.
Detection of Nuclear Chromatin Condensation
The
fluorescent dye,
2`-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5`-bi-1H-benzimidazole
trihydrochloride pentahydrate (bisbenzimide, Hoechst 33258; Molecular
Probes, Eugene, OR) was used to visualize the morphological features of
nuclei in chick neuronal cultures. Cells grown on glass coverslips were
fixed with 4% paraformaldehyde in PBS overnight at 4 °C, treated
with bisbenzimide (1 µg/ml) in PBS for 15 min at 22 °C, and
washed twice with PBS. Coverslips were mounted with an anti-bleaching
fluorescence medium and viewed under UV light using a Zeiss Axiophot
fluorescence microscope.Immunohistochemical Detection of
Apoptosis
Detection of apoptosis was performed by direct
immunofluorescence staining using fluorescein isothiocyanate-conjugated
anti-digoxigenin-labeled nucleotides incorporated into DNA fragments by
3`-OH end extension (TUNEL) using an ``Apotag'' Kit (Oncor
Inc.). Neuronal cultures on glass coverslips were fixed with 1%
paraformaldehyde in PBS for 10 min at 22 °C. The coverslips were
washed twice with PBS (5 min each) and subsequently treated according
to protocols provided by Oncor. Cells were counter-stained by mounting
the coverslips with propidium iodide (0.5 µg/ml) in anti-bleaching
fluorescence mounting medium and viewed as described above.Uptake and Retention of
[
Freshly plated neurons
were used about 1 h after they were firmly attached. 2 µCi of
[H]2-Deoxyadenosine and
[
H]Adenosine
H]dAdo (specific activity 28.4 Ci/mmol, Moravek
Biochemicals, Brea, CA) was directly added to the medium (1 ml) and
dishes were maintained at 37 °C in a CO
incubator for
various times. Cells were pretreated with inhibitors (3 µM dilazep or 10 µM nitrobenzyl thioinosine (NBTI)) for
30 min prior to addition of [
H]dAdo. For
competition studies, 100 µM unlabeled dAdo or adenosine
was added with [
H]dAdo. For temperature studies,
dishes were placed on ice for 15 min and then
[
H]dAdo added to study uptake at 4 °C. After
each specified time point, cells were washed 3-4 times with
ice-cold Krebs solution and finally extracted in 1.0 ml of ice-cold
0.6% trichloroacetic acid. After centrifugation, a portion of the
supernatant was used for measurement of total radioactivity
([
H]dAdo plus
H-labeled metabolites)
in a scintillation counter and the pellet was saved for protein
analysis. Radioactivity retained by the neurons during each incubation
was expressed as cpm/µg protein. Uptake and retention of
[
H]adenosine was measured similarly, using 2
µCi of [
H]adenosine/ml of medium (specific
activity 30.4 Ci/mmol).
Analysis of dATP Pool in Neurons
dATP content was
determined using the synthesis of radiolabeled poly(dA-T) with dATP as
the limiting factor(18) . Neurons from 2 culture dishes were
extracted on ice in 250 µl of 0.4% perchloric acid. Extracts were
neutralized with KOH and precipitate removed by centrifugation. A
portion of the extract was used for protein determination. 100 µl
of extract (or dATP standard) was added to 100 µl of reaction
mixture (200 mM glycine-NaOH, pH 9.2, 20 mM MgCl, 100 µM TTP, 0.075 units of
polydeoxyadenylic-thymidilic acid, and 0.0025 µCi of
[
H]TTP (specific activity 103.9 Ci/mmol, DuPont
NEN). The reaction was started by addition of 100 µl (1 unit) of
DNA polymerase and all tubes incubated for 1 h at 37 °C. The
reaction was stopped and poly(dA-T) precipitated by addition of 5 ml of
ice-cold 10% trichloroacetic acid. Precipitate was collected by vacuum
filtration (0.45-µm filters). Filters were washed 4 times with
about 2 ml of 5% trichloroacetic acid and transferred to vials for
scintillation counting. Concentration of dATP in the sample was
determined from a standard curve of counts/min from reactions run with
known concentrations of dATP.
Protein Estimation
The protein pellet was
dissolved in 100 µl of 1 N NaOH for protein estimation as
described previously(19) .Statistical Analysis
In all cases, cultures
treated with dAdo and/or other agents were compared to matched,
untreated control cultures. Data are presented as means ± S.E.
and differences compared using Student's t test (two
group comparisons) with p < 0.01 significant.
H]dAdo in freshly plated neurons over the time
indicated. C, effects of 3 µM dilazep (hatchedbar), 10 µM NBTI (cross-hatchedbar), 4 °C (openbar) and competition by 100 µM unlabeled dAdo (shadedbar) and adenosine (filledbar) as a percentage of
[
H]dAdo uptake in control cultures. Values
represent the mean (± S.E.) of 4-6 experiments. *
represents p < 0.01 compared to untreated controls,
Student's t test.
H]dAdo (2 µCi/ml medium, specific activity
28.4 Ci/mmol), retention of the label increased to a maximum by 10 min
and remained near that level over the next 5 h (Fig. 3B). The uptake was remarkably less when compared
to the accumulation of [
H]adenosine (2 µCi/ml
medium, specific activity 30.4 Ci/mmol) after a 100-min incubation
(3260 ± 849 versus 12470 ± 3200 cpm/µg
protein, n = 10). Furthermore, classic inhibitors of
the nucleoside transporter NBTI (10 µM) or dilazep (3
µM) blocked [
H]adenosine uptake by
87 ± 4% (n = 4), but had little or no effect on
[
H]dAdo uptake (Fig. 3C).
Unlabeled dAdo or adenosine (100 µM) caused only partial
inhibition of [
H]dAdo uptake, and the uptake of
[
H]dAdo was not sensitive to temperature. These
results suggest that dAdo enters sympathetic neurons primarily by a
passive process rather than by the nucleoside transporter.
H]dAdo accumulation in
sympathetic neurons (not shown).
H]dAdo was also unaffected by these agents.
Furthermore, [
H]dAdo uptake was substantially
lower than that of [
H]adenosine, only slightly
affected by competition with unlabeled adenosine or dAdo, and
insensitive to temperature, supporting the idea that dAdo is not
carried by the classic nucleoside transporter. Finding that adenosine
but not dAdo accumulates in sympathetic neurons via an NBTI sensitive
transporter is distinctly different from nucleoside transport
properties in other cell types where adenosine and dAdo enter via a
similar mechanism. Although at least five different nucleoside
transporters have been identified in mammalian cells(29) , the
kinetics and inhibitor sensitivity of transport have been reported to
be the same for adenosine and dAdo in a variety of systems, including
leukemia cell lines(30, 31) , human
erythrocytes(32) , and fibroblast lysosomes (33) .
100 µM, Fig. 5B) and that needed to produce maximum cell death
(
300 µM, Fig. 1A). This, plus the
finding that dATP levels increased almost immediately while cell death
took 24-48 h, supports the idea that factors other than dATP may
be involved in dAdo toxicity. Studies are in progress to determine the
chronological order of cell growth arrest, ATP and dATP content, RNA
and protein synthesis, and changes in the mRNA levels of protooncogenes
considered to play a role in neuronal apoptosis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.