©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Deoxynucleoside Induces Neuronal Apoptosis Independent of Neurotrophic Factors (*)

Arun R. Wakade Dennis A. Przywara (§) Kenneth C. Palmer (1) Jayant S. Kulkarni Taruna D. Wakade

From the Departments of Pharmacology and Pathology, School of Medicine, Wayne State University, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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, 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.


INTRODUCTION

2`-Deoxyadenosine (dAdo)() 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) .

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 -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.


MATERIALS AND METHODS

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 [H]2-Deoxyadenosine and [H]Adenosine

Freshly plated neurons were used about 1 h after they were firmly attached. 2 µCi of [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.


RESULTS

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 [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.



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 [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.

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 [H]dAdo accumulation in sympathetic neurons (not shown).

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).




DISCUSSION

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 [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) .

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 (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.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 Pharmacology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201.

The abbreviations used are: dAdo, 2-deoxyadenosine; SCIDS, severe combined immunodeficiency syndrome; ADA, adenosine deaminase; NGF, nerve growth factor; PBS, phosphate-buffered saline; NBTI, nitrobenzyl thioinosine; ITu, 5`-iodotubercidin.


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