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J Biol Chem, Vol. 273, Issue 45, 29626-29634, November 6, 1998
From the Although purinergic compounds are widely involved
in the intra- and intercellular communication of the nervous system,
little is known of their involvement in the growth and regeneration of neuronal connections. In dissociated cultures, the addition of adenosine or guanosine in the low micromolar range induced goldfish retinal ganglion cells to extend lengthy neurites and express the
growth-associated protein GAP-43. These effects were highly specific
and did not reflect conversion of the nucleosides to their nucleotide
derivatives; pyrimidines, purine nucleotides, and membrane-permeable,
nonhydrolyzable cyclic nucleotide analogs were all inactive. The
activity of adenosine required its conversion to inosine, because
inhibitors of adenosine deaminase rendered adenosine inactive.
Exogenously applied inosine and guanosine act directly upon an
intracellular target, which may coincide with a kinase described in
PC12 cells. In support of this, the effects of the purine nucleosides
were blocked with purine transport inhibitors and were inhibited
competitively with the purine analog 6-thioguanine (6-TG). In PC12
cells, others have shown that 6-TG blocks nerve growth factor-induced
neurite outgrowth and selectively inhibits the activity of protein
kinase N, a partially characterized, nerve growth factor-inducible
serine-threonine kinase. In both goldfish and rat retinal ganglion
cells, 6-TG completely blocked outgrowth induced by other growth
factors, and this inhibition was reversed with inosine. These results
suggest that axon outgrowth in central nervous system neurons
critically involves an intracellular purine-sensitive mechanism.
Under normal circumstances, neurons in the adult mammalian central
nervous system fail to regenerate axons injured by stroke or trauma,
resulting in long lasting disabilities in sensory, motor, or cognitive
functions. However, many central nervous system neurons have the
potential to regrow their axons if exposed to appropriate growth
factors and/or if inhibitory influences that normally prevail in the
mature central nervous system are suppressed (1-3). To identify
factors that foster axonal regrowth in the central nervous system, we
have turned to the classic model of optic nerve regeneration in lower
vertebrates. Following injury to the optic nerve, retinal ganglion
cells (RGCs)1 in fish and
amphibia re-extend axons that synapse upon appropriate central targets
within 1-2 months (4). In a dissociated cell culture model of this
system, two factors secreted by optic nerve glia were found to be
responsible for inducing axon outgrowth from goldfish RGCs (5, 6). The
more potent of these, axogenesis factor-1 (AF-1), has a molecular mass
<1 kDa and stimulates lengthy axonal outgrowth without altering cell
survival. AF-2 is a larger polypeptide that exerts a significant but
less pronounced effect than AF-1. The specificity of these effects is
demonstrated by the inactivity of a host of polypeptide growth factors
or low molecular mass differentiation factors in this system (5).
In the present study, we have investigated the role of purines in the
regeneration of RGC axons. Although the involvement of purine
nucleosides in the intra- and intercellular signaling of the nervous
system is well established, their role in axonal outgrowth has not been
investigated extensively. Extracellularly, many nerve terminals release
adenosine or ATP as cotransmitters that act via P1 or
P2 purinergic receptors to mediate changes in ion fluxes
and other physiological effects in target cells (7-11). In PC12
pheochromocytoma cells, purines partially mimic or augment the effects
of nerve growth factor (12), whereas in sympathetic neurons, adenosine
inhibits neurite outgrowth and induces apoptotic cell death (13). We
show here that, in dissociated retinal ganglion cells, inosine and to a
lesser extent guanosine act directly upon an intracellular pathway to
stimulate axonal outgrowth and expression of the growth-associated
protein GAP-43, whereas the purine analog 6-thioguanine acts
competitively with these purines to arrest growth.
Dissociated Retinal Cultures--
Goldfish (Comet Variety, Mt.
Parnell Fisheries, Mt. Parnell, PA), 6-10 cm in length, were
dark-adapted, and their retinas were dissected. Retinas were incubated
with papain (20 µg/ml) activated with cysteine (2.8 mM)
for 30 min at room temperature, and then dissociated by gentle
trituration. Repeated cycles of trituration and sedimentation yielded
cultures nearly homogeneous in ganglion cells, which are readily
identified by their oval shape, phase-bright appearance, size
(diameter = 12-25 µm), and extension of 1 or 2 neurites of
uniform caliber; these criteria have been verified by retrograde
labeling (5, 14). Low density cultures were obtained by plating
~5 × 103 cells/well in poly-L-lysine
coated, 24-well culture dishes (Costar, Cambridge, MA). Cells were
maintained at 21 °C in a serum-free, defined medium as described
(5, 6).
Dissociated cultures of purified rat retinal ganglion cells were
prepared by immunopanning (15). Briefly, retinas from postnatal day 8 Sprague-Dawley rats were dissociated enzymatically, and macrophages
were removed by incubation with an anti-rat macrophage antibody
(Accurate) followed by immunopanning with an anti-rabbit IgG antibody.
Ganglion cells were separated from non-adhering cells by immunopanning
with an anti-Thy-1 antibody (American Type Culture Collection) and then
dislodged with trypsin. RGCs of >95% purity were maintained at
37 °C in a 5% CO2 incubator in the same medium used for
goldfish RGCs except for the addition of 5 µM forskolin
to enhance cell viability (16) and 26 mM bicarbonate. Based
upon other studies (17), stimulation of growth by recombinant rat
ciliary neurotrophic factor (CNTF; 20 ng/ml, Promega, Madison, WI) was
used as a positive control.
Experimental Design--
Samples were plated in quadruplicate in
randomized positions of a 24-well plate. To ensure that scoring was
conducted blind, the code was concealed until experiments were
completed. All experiments included 4 wells of a negative (defined
medium only) and a positive control (partially purified AF-1, 20-30%
concentration). Growth and survival were assessed after 6 days by
examining 15-25 consecutive fields of each well using phase contrast
microscopy (at 400× magnification, ~150 ganglion cells/well).
Extension of a process at least 5 cell diameters in length was used as
our criterion for outgrowth (5). Means and standard errors were
calculated (Cricket Graph, CA Associates, Islandia, NY), and data were
normalized by subtracting the growth in the negative controls (usually
4-5%) and dividing by the net growth in the positive controls. In the
most favorable experiments, >50% of RGCs exposed to AF-1 for 6 days
extended axons at least 5 cell diameters in length, and many were
several hundred microns in length. At longer intervals, almost all
AF-1-treated ganglion cells extend lengthy
axons.2 Group comparisons
reported here are based upon pairwise, two-tailed Student's
t tests. Several independent experiments were performed for
all samples. In some cases, cell viability was assessed using the vital
dye 5,6-carboxyfluorescein diacetate. For this, medium was replaced
just prior to counting with a 0.005% (w/v) solution of
carboxyfluorescein diacetate in PBS for 5 min. Cells were rinsed with
PBS, and the fluorescent reaction product was visualized under UV
illumination. Survival is reported as the number of viable RGCs/400×
field. Experiments using rat RGCs were carried out in a similar fashion
except that cells were fixed and counted after 2 days. Outgrowth is
reported as the fold increase in growth relative to negative controls.
Samples--
Adenosine, AMP, adenosine deaminase (ADA), ADP,
ATP, 8-bromo 3',5'-cyclic guanosine monophosphate, cAMP, cGMP,
cytidine, guanosine, hypoxanthine, inosine, 5'-inosine monophosphate,
GAP-43 Immunostaining--
Recombinant GAP-43 was made by
transforming Escherichia coli with a zebrafish cDNA
isolated by Dr. Eva Reinhard (18) subcloned into the prokaryotic
expression vector pTrcHisB (Invitrogen). The protein was purified by
Ni2+-nitrilotriacetic acid affinity chromatography and used
to immunize rabbits. Prior to use, nonspecific binding of the antiserum
was reduced by diluting it 1:1000 in 50 mM PBS containing
1% bovine serum albumin (Sigma) and reacting it at 4 °C with a
1 × 14-cm strip of a Western blot on nitrocellulose membrane
containing ~200 µg of proteins from goldfish internal organs
separated by SDS-polyacrylamide gel electrophoresis. The specificity of
the pre-adsorbed antibody is demonstrated on Western blots, where it
reacts only with a 48-kDa band that is enriched in retinal ganglion
cells undergoing regeneration (see below). Immunostaining was carried
out in cultured neurons fixed in methanol (10 min, 4 °C) and reacted
overnight (4 °C) with the immune serum prepared as described above
or with non-immune serum. Cells were washed with PBS (3 × 10 min), reacted with fluorescein isothiocyanate-conjugated anti-rabbit
IgG (Jackson Immuno Research; 1:500 in PBS with 1% bovine serum
albumin), and washed three times. For Western blots, ~106
goldfish retinal cells were grown in 100-mm dishes in control medium,
AF-1 (30%), or inosine (50 µM) for 6 days. Cells were collected and centrifuged, and 50 µg of protein from each was separated by SDS-polyacrylamide gel electrophoresis (14% acrylamide), transferred to polyvinylidene difluoride membrane (200 mA, 15 h,
4 °C), and probed for GAP-43 in a procedure similar to that used for
immunocytochemistry except for the use of a horseradish peroxidase-conjugated secondary antibody followed by ECL reagent (Amersham Pharmacia Biotech).
Purines Nucleosides Stimulate Axonal Outgrowth from Goldfish
Retinal Ganglion Cells--
In agreement with previous findings (5,
6), AF-1, a low molecular mass growth factor secreted by optic nerve
glia, stimulated extensive outgrowth from goldfish retinal ganglion
cells (Fig. 1a); controls
maintained in the absence of AF-1 remained quiescent (Fig.
1b). At 100 µM, adenosine and guanosine
stimulated considerable outgrowth (Fig. 1c), whereas the
pyrimidines cytidine, uridine, and thymidine had no activity. These
results are shown quantitatively in Fig.
2a, with data normalized by
the net growth in AF-1-treated positive controls. A more complete
dose-response curve (Fig. 2b) shows that at 50-100
µM adenosine induces a response 60% of that stimulated
by AF-1 but that outgrowth decreases at higher concentrations. Based
upon 10 independent experiments, the EC50 for adenosine was
calculated to be 11.7 ± 1.3 µM (mean ± S.E.).
Guanosine was less potent (EC50 = 32.4 ± 13.2 µM, based upon five experiments), but at concentrations
above 100 µM, it stimulated the same maximal level of
outgrowth as adenosine, with no decrease in activity at higher
concentrations. None of these agents altered cell survival (see below,
Fig. 3c).
Purine Nucleotides Are Less Active than Nucleosides--
cAMP and
ATP at concentrations of 1-100 µM had no effect on
outgrowth (Fig. 2c). AMP and ADP were both inactive at 1 and
10 µM but showed slight activity at 100 µM
(p = 0.05). These results make it unlikely that the
purine nucleosides are acting as precursors to nucleotides that
stimulate P2 purinergic receptors (10, 11).
Another possibility is that the purines could serve as precursors for
cyclic nucleotides that act as second messengers to stimulate
outgrowth. However, nonhydrolyzable, membrane-permeable analogs of cAMP
(8-bromoadenosine-3',5' cyclic monophosphorothioate) and of cGMP
(8-(4-chlorophenylthio) guanosine-3',5'-cyclic monophosphate) were
inactive even at concentrations up to 1 mM (Fig.
2d). Two other cyclic nucleotide analogs, dibutyryl cAMP and
8-bromo-cGMP, likewise showed no activity (Fig. 2d). As with
the nucleosides and other nucleotides, none of the cyclic nucleotides
affected cell survival (data not shown).
Blockade of Adenosine Receptors--
The two most common adenosine
receptors (A1 and A2) are effectively blocked
with 20 µM 8-p-sulfophenyl-theophylline (19, 20). However, this concentration of
8-p-sulfophenyl-theophylline had no effect on outgrowth
stimulated by adenosine or guanosine (Fig. 3a, lanes
4 and 6). A further indication that adenosine is not
inducing growth through extracellular adenosine receptors comes from
studies using the non-hydrolyzable analog, 2-CA, which acts as an
agonist at the A1, A2, and A3
receptors (21). At 10 and 100 µM, 2-CA actually caused
growth to decline below base line (Fig. 3a, lane
7; decline significant at p < 0.001).
Adenosine Must Be Hydrolyzed to Stimulate Outgrowth--
The
previous results indicate that adenosine must be metabolized to be
active. Adenosine is converted to inosine by ADA (22), and we therefore
investigated the effects of inhibiting this enzyme with either DCF (10 µM) or erythro-9-(2-hydroxy-3-nonyl) adenine (10 µM). In the presence of DCF, 100 µM
adenosine caused growth to decline below base line (Fig. 3b,
lane 5 versus lane 4: p < 0.01) and
impaired cell survival (Fig. 3c, lane 5 versus lane 4: p < 0.001). These effects can be attributed
specifically to nonhydrolyzed adenosine and did not occur when DCF was
used alone, with AF-1, or with guanosine (Fig. 3, b and
c, lanes 2 and 8). Like DCF,
erythro-9-(2-hydroxy-3-nonyl) adenine prevented 100 µM
adenosine from stimulating growth and caused cell survival to decline
by 30% (data not shown). Further evidence that the effects of
adenosine require its hydrolysis comes from experiments in which we
added exogenous ADA. At 0.4 unit/ml, ADA failed to diminish axon growth
stimulated by 100 µM adenosine (Fig. 3b, lane 6) and had no effect on survival.
Inosine Is the Active Metabolite--
As anticipated, inosine, the
primary product of adenosine deamination, was a potent activator of
axon outgrowth. The EC50 for inosine was estimated to be
11.4 ± 1.4 µM (average from seven experiments), and
a maximal response equal to about 60% the level stimulated by AF-1 was
attained at concentrations of 50 µM (Fig. 4a). Unlike adenosine,
increasing the concentration of inosine up to 1 mM did not
cause growth to decline. Metabolic hydrolysis of inosine yields
hypoxanthine, which was inactive. Inosine 5' monophosphate had no
effect at 10 µM but showed modest activity at 100 µM (Fig. 4a).
Purine Nucleosides Stimulate Growth through an Intracellular
Pathway--
Two inhibitors of purine transport, NBTI and
dipyridamole, were used to investigate whether inosine and guanosine
must be transported into neurons to stimulate growth. At 20 µM, NBTI prevented both nucleosides from stimulating
outgrowth (Fig. 4b, lanes 4 and 6 versus
lanes 3 and 5, respectively; p < 0.01)
but had little effect on AF-1-induced growth (lanes 1, 2).
Dipyridamole (10 µM) likewise blocked the effects of
inosine completely (p < 0.01; guanosine not tested).
Effect of Cell Density--
The cultures used here contain
70-90% ganglion cells, with the remainder representing other neural
and non-neuronal elements of the retina (5, 14). This heterogeneity
raises the possibility that inosine or guanosine could act upon another
cell type that secretes a secondary factor that stimulates RGCs. In
this case, the effect of the purines would vary with cell density,
because the concentration of any secondary factor should decrease as
cell density declines. However, the level of outgrowth stimulated by either 100 µM inosine or 100 µM guanosine
remained unchanged over a 10-fold density range, down to 5 cells/mm2 (data not shown).
GAP-43 Expression--
A hallmark of optic nerve regeneration
in vivo is the dramatic up-regulation in expression of the
phosphoprotein, GAP-43 (23-25). To investigate whether purine
nucleoside stimulation mimics this up-regulation, we carried out
immunohistochemistry using an antiserum against zebrafish GAP-43. As
shown in Fig. 5 (c,
e, and g), AF-1, inosine, and guanosine all
dramatically altered GAP-43 expression relative to negative controls.
This is also evident on Western blots (5 h). In a semi-quantitative
analysis, we rated the level of GAP-43 immunoreactivity from 0 (none)
to 3 (intense) and correlated staining intensity with the length of the
axon of the cell (150-200 cells/condition). Inosine produced a highly
significant increase in the number of cells with intensity >1
( Blockade of Axonal Outgrowth with 6-TG--
In PC12 cells and
peripheral neurons, the purine analog 6-TG selectively blocks
NGF-induced differentiation but not survival (26, 27). Likewise, 6-TG
(10 µM) completely blocked AF-1-stimulated outgrowth in
goldfish RGCs (Figs. 1d and 6a, lane
2; p < 0.001) with no effect on cell survival
(Fig. 6b, lane 2).
This concentration of 6-TG reduced outgrowth stimulated by 25 µM inosine by only 50% (Fig. 6a, lanes
4 versus lane 3) and had no effect on growth stimulated by 100 µM inosine (or 100 µM guanosine; Figs.
1g and 6a, lanes 5-8). At 100 µM, inosine restored the growth induced by AF-1 in the
presence of 10 µM 6-TG back to its original level (Figs.
1h and Fig. 6a, lane 10 versus
lane 9), which is significantly above the level induced by
inosine alone (Fig. 6a, lane 10 versus lane 6,
p < 0.01). In additional studies, higher
concentrations of 6-TG (200 µM) completely blocked growth
induced by 50 µM inosine, whereas decreasing
concentrations of the inhibitor had progressively less of an effect
(data not shown). Thus, inosine and 6-TG appear to compete for the same
target molecule, and both of these modulate the effects of the
endogenous growth factor AF-1.
Because 6-TG has a free thiol, it could be acting by nonspecific redox
effects rather than as a purine analog. To test this possibility, we
used a free radical scavenger, Mammalian Retinal Ganglion Cells--
Retinal ganglion cells,
purified to homogeneity from 8-day-old rats, showed a modest level of
spontaneous growth when grown in defined medium for 2 days (Fig.
7a). Inosine at 25 µM stimulated a 52% increase in outgrowth
(p < 0.002 compared with controls; Fig. 7,
b and c, lane 2) but failed to alter
cell survival (Fig. 7d). A similar effect was seen with 50 µM inosine, whereas 10 µM was ineffective
(not shown). CNTF induced a 3-fold increase in outgrowth over control
levels (Fig. 7c, lane 3; cf. Ref. 17) and enhanced survival significantly. 10 µM 6-TG blocked
CNTF-induced outgrowth but did not prevent CNTF from enhancing cell
survival (Fig. 7, c and d, lane 5).
The addition of 50 µM inosine restored a high level of
outgrowth to CNTF-treated cells exposed to 6-TG (Fig. 7c,
lane 6).
Mitogen-activated Protein Kinase and PI 3-Kinase Pathways--
In
PC12 cells, activation of MEK-1 and/or MEK-2 is necessary and
sufficient to induce neurite outgrowth (28, 29). Conversely, PD 098059, which acts as a highly specific inhibitor of MEK-1/-2 activation (30,
32), blocks NGF-induced differentiation of PC12 cells (31). At 50 µM, PD 098059 blocked ~50% of the outgrowth induced by
100 µM inosine (p < 0.001) but did not
alter the effects of AF-1 (Fig. 8;
similar results obtained in five separate experiments). PD 098059 at 25 µM was equally effective in blocking growth stimulated by
inosine, whereas at 100 µM, it had a stronger effect on
growth but also affected cell survival (data not shown). NGF signaling in PC12 cells also activates PI 3-kinase (33), and inhibitors of this
pathway also affect differentiation (34). LY 294002, a potent
phosphatidylinositol 3-kinase inhibitor (35), showed similar effects as
PD 098059, inhibiting about half the activity of 100 µM
inosine (p < 0.002) without affecting growth
stimulated by AF-1 (Fig. 8). The combination of PD 098059 plus LY
294002 completely blocked outgrowth stimulated by inosine
(p < 0.001) but only partially diminished growth
stimulated by AF-1 (loss significant at p < 0.002).
These results show that the purine nucleosides inosine and
guanosine act through an intracellular mechanism to stimulate axonal outgrowth and GAP-43 expression in retinal ganglion cells. Inosine is
the more potent of the two, perhaps because it preferentially activates
one or more intracellular target(s) or perhaps because it is more
readily converted to another active metabolite. It is clear that the
nucleosides are not acting primarily as precursors to the cyclic
nucleotides, however, because membrane-permeant, nonhydrolyzable
analogs of cAMP and cGMP were inactive. The principle product of
inosine hydrolysis, hypoxanthine, was likewise ineffective in causing
growth, and hence the biologically active agent may be inosine
per se. In other cell types, inosine acting through an
intracellular mechanism has been shown to prevent cell death in
astrocytes (36) and to evoke relaxation of the aorta (37). In addition,
inosine was shown to be the principal factor in liver conditioned
medium that stimulates differentiation in chick sympathetic neurons
(38).
The intracellular target of inosine may coincide with a
purine-sensitive kinase that has been implicated in mediating
NGF-induced differentiation. In PC12 cells and in sensory and
sympathetic neurons, the purine analog 6-TG blocks NGF-induced neurite
outgrowth without affecting survival (26, 27). Research into the
mechanism of action of 6-TG has shown that it selectively inhibits an
NGF-activated, 45-47-kDa serine-threonine kinase, protein kinase N
(39, 40), that remains to be characterized further. In the present
study, a competition between inosine and 6-TG was evident; whereas
outgrowth stimulated by a nearly saturating level of inosine was
partially blocked by 6-TG at 10 µM, outgrowth induced by
higher concentrations of inosine (or guanosine) was not. However,
increasing the concentration of 6-TG progressively inhibited the
activity of higher concentrations of inosine. Hence, inosine may be
acting as an agonist at the same site at which 6-TG acts as an
antagonist, i.e. protein kinase N. The structural
similarities among inosine, guanosine, and 6-TG would be consistent
with a common site of action.
Growth induced by the endogenous low molecular mass factor AF-1 was
more robust than by inosine and was more resistant to disruption by
inhibitors of MEK-1/-2 and PI 3-kinase. Growth induced by inosine was
reduced by half with the MEK-1/-2 inhibitor PD 098059 or by the PI
3-kinase inhibitor LY 294002 and was blocked completely by the
combination of the two. In contrast, growth induced by AF-1 was
unaffected by either inhibitor alone and still remained high in the
presence of the two combined. On the other hand, growth stimulated by
AF-1 was completely blocked by 6-TG, whereas inosine seemed to work
competitively with 6-TG and restored AF-1-activated growth back to its
original level in the presence of the inhibitor. One interpretation of
these results is that growth stimulated by AF-1 and by inosine is
transduced through protein kinase N but that AF-1 activates additional
downstream pathways besides MEK-1/-2 and PI 3-kinase. Axon outgrowth in
mammalian RGCs also appears to utilizes a purine-sensitive mechanism,
because inosine stimulated some degree of outgrowth by itself and
reversed the inhibition of CNTF-induced outgrowth by 6-TG. Throughout
these studies, AF-1, inosine, nor 6-TG had any effect on cell survival. A similar dissociation between intracellular signaling pathways leading
to survival versus outgrowth is well documented for other cell types (27, 29, 30).
In vivo, optic nerve regeneration is marked by an increase
in ganglion cells' expression of the membrane phosphoprotein GAP-43 (23, 24, 25, 41). In goldfish RGCs, purines and AF-1 both stimulated
GAP-43 expression, thus mimicking at least one aspect of the molecular
changes that underlie axonal regeneration in vivo. In
preliminary studies, inosine also strongly stimulated promoter activity
for Adenosine was active only when hydrolyzed to inosine. The
nonhydrolyzable adenosine analog 2-CA failed to induce growth, whereas inhibiting the hydrolysis of adenosine itself led to diminished cell
survival. Thus, the failure of adenosine to inhibit growth may be
secondary to its effects on cell survival. Adenosine has previously
been shown to induce apoptotic cell death in sensory neurons (13) and
to inhibit in vitro regeneration of the frog sciatic nerve
(42). In PC12 cells, adenosine and its nucleotide derivatives
potentiated NGF-induced outgrowth (12). However, because inosine also
had a positive effect in that study and because adenosine deaminase was
not inhibited, it is possible that the stimulatory effects of the
adenosine compounds all ultimately reflect the action of inosine.
Positive effects of guanosine on neurite outgrowth in PC12 cells have
also been reported recently (43). In the present study, pyrimidine
nucleosides had no effect on outgrowth, whereas the purine nucleotides
showed only low levels of activity. Catabolic enzymes that rapidly
convert cAMP, ATP, ADP, and AMP to adenosine are present
extracellularly in cultured neurons (12, 44-46), and the low levels of
activity found for 5' inosine monophosphate, 5' AMP, and ADP may
therefore reflect their conversion to inosine. Adenosine, inosine, and
guanosine coexist intracellularly, and it is even possible that purine
nucleosides might act as second messengers in regulating the neuron's
growth state. It will be of interest to identify the intracellular
target(s) of the purines, whether it is protein kinase N or another
molecule, and to characterize in detail the pathways that lead to axon growth.
We thank Dr. Gui-lan Yao, Eli Diamond, and
Eunice Wang for technical assistance, Ute Laessing for the anti-GAP-43
antibody, and Drs. Lloyd Greene (Columbia University College of
Physicians and Surgeons) and Robert Greene (Brockton Veterans Affairs
Hospital and Harvard Medical School) for helpful discussions.
*
This work was provided by the National Eye Institute (R01 EY
05690), Boston Life Sciences, Inc., and the Boston Neurosurgical Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Labs. for
Neuroscience Research in Neurosurgery, Children's Hospital, 300 Longwood Ave., Boston MA 02115. Tel.: 617-355-6368: Fax: 617-730-0636; E-mail: benowitz{at}a1.tch.harvard.edu.
The abbreviations used are:
RGC, retinal
ganglion cell; AF-1, axogenesis factor-1; CNTF, ciliary neurotrophic
factor; PBS, phosphate-buffered saline; ADA, adenosine deaminase; 6-TG, 6-thioguanine; DCF, deoxycoformycin; 2-CA, 2-chloroadenosine; NBTI, 4-(nitrobenzyl-6-thioinosine); MEK, mitogen-activated protein kinase
kinase; PI, phosphatidylinositol; NGF, nerve growth factor.
2
B. Petrausch and L. Benowitz, unpublished data.
3
N. Irwin, A. Taghinia, D. Goldman, and L. Benowitz, unpublished data.
4
R. Petrausch, C. Stuermer, and L. Benowitz,
unpublished data.
Axon Outgrowth Is Regulated by an Intracellular Purine-sensitive
Mechanism in Retinal Ganglion Cells*
§¶,,,§,
,,§
Laboratories for Neuroscience Research in
Neurosurgery, Department of Biology,
University of Konstanz, Konstanz, D-78434, Germany
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-tocopherol, 6-thioguanine (6-TG), thymidine, and uridine were from
Sigma. 8-p-Sulfophenyl-theophylline, dibutyryl cAMP, and
deoxycoformycin (DCF) were from Calbiochem; 2-chloroadenosine (2-CA)
and erythro-9-(2-hydroxy-3-nonyl) adenine were from Research
Biochemicals, Inc. (Natick, MA); and 4-(nitrobenzyl-6-thioinosine)
(NBTI) was from Aldrich. Membrane-permeable, nonhydrolyzable analogs of
cAMP and of cGMP were from Biolog. The specific inhibitor to
mitogen-activated protein kinase kinase-1 and -2 (MEK-1 and -2), PD
098059, and the inhibitor to phosphatidylinositol (PI) 3-kinase, LY
294002, were from Sigma. In experiments using these, RGCs were
pretreated with inhibitors overnight prior to introducing growth
factors. Thereafter, half the medium was changed daily for 5 days to
maintain high concentrations of growth factors and inhibitors. Because
PD 098059 and LY 294002 were prepared in Me2SO, positive
and negative controls contained Me2SO at the concentration
required to solubilize the drugs (0.6% final).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (19K):
[in a new window]
Fig. 1.
Response of goldfish retinal ganglion cells
to purine nucleosides and inhibitors. Dissociated RGCs were
maintained in defined medium for 6 days as described under
"Experimental Procedures." a, positive controls treated
with AF-1. b, negative controls with defined media alone.
c and d, outgrowth in response to guanosine
(c) and inosine (d), each at 100 µM. e, the purine transport inhibitor NBTI (20 µM) blocks the effect of 100 µM inosine.
f and g, the purine analog 6-TG (10 µM) completely inhibits outgrowth stimulated by AF-1
(f) but is ineffective in the presence of high
concentrations of inosine (100 µM; g).
h, 100 µM inosine restores outgrowth of cells
treated with AF-1 plus 10 µM 6-TG to its original level.
Cells were stained with the vital dye 5,6-carboxyfluorescein diacetate
and visualized under UV illumination. Scale bar (shown in
a) is 50 µM.
View larger version (24K):
[in a new window]
Fig. 2.
Effects of nucleosides and nucleotides on
axonal outgrowth: quantitation. a, the nucleosides
adenosine (Ado), guanosine (Guo), cytidine
(Cyt), uridine (Uri), and thymidine
(Thy) were bioassayed at 1, 10, and 100 µM.
Data were normalized by subtracting the level of growth in negative
controls and then dividing by the net growth in positive controls
(AF-1, 25% concentration). b, dose-response curves for
adenosine and guanosine. EC50 values estimated from these
and other experiments are 11.4 ± 1.3 µM for
adenosine and 32.4 ± 13.2 µM for guanosine
(mean ± S.E.). c, effects of adenosine nucleotides.
d, effects of membrane-permeable analogs of cyclic AMP
(dibutyryl cAMP (dB cAMP) and 8-bromoadenosine-3',5' cyclic
monophosphorothioate (Sp-8Br cAMPs)) or cyclic GMP (8-bromo
cyclic GMP (8-Br cGMP) and 8-(4-chlorophenylthio)
guanosine-3',5'-cyclic monophosphate (8-pcpt-cGMP). Data
represent the means ± S.E. (not shown if <0.02) and are pooled
from two to four independent experiments. p values are based
upon two-tailed t tests comparing growth in the sample with
that of the negative control. *, p < 0.05; **,
p < 0.01; ***, p < 0.001.
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Fig. 3.
Effects of adenosine receptor agonists,
antagonists, and metabolic inhibitors. a, adenosine
does not stimulate growth via extracellular receptors. Lanes
1-6, outgrowth stimulated by AF-1, 100 µM adenosine
(Ado), or 100 µM guanosine (Guo) is
unaffected by the addition of 20 µM
8-p-sulfophenyl-theophylline (8-PST), an
inhibitor of A1 and A2 adenosine receptors
(compare growth in lane 1 versus lane 2, lane 3 versus
lane 4, and lane 5 versus lane 6). Lane 7,
the nonhydrolyzable adenosine analog 2-CA (100 µM)
diminishes outgrowth below base-line levels (p < 0.001 in three experiments). b and c, effects of DCF
and exogenous ADA on outgrowth and survival. Whereas augmenting
adenosine hydrolysis with exogenous ADA leaves the activity of
adenosine unaltered (b, lane 6), blocking
endogenous ADA activity with DCF causes adenosine to suppress growth
(b, lane 5) and survival (c,
lane 5). The effect of ADA on guanosine was not tested. ***,
p < 0.001.
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Fig. 4.
Inosine stimulates axonal outgrowth through
an intracellular mechanism. a, at concentrations above
50 µM, inosine stimulates about 60% the maximal level of
growth activated by AF-1; its EC50 is estimated to be
11.4 ± 1.4 µM. Hypoxanthine is inactive, whereas 5'
inosine monophosphate (IMP) is an order of magnitude less
active than inosine. b, at 20 µM, NBTI, an
inhibitor of purine transport, prevents inosine (ino) or
guanosine (guo) from stimulating growth (100 µM) but does not alter the effects of AF-1. ***,
differences significant at p < 0.001.
2 = 18.4; p < 0.0001), and the
intensity of GAP-43 immunostaining was correlated with axon length
(r = 0.544; p < 0.0001). Appreciable levels of immunostaining were seen in 100% of inosine-treated cells
having axons >5 cell diameters in length but in only 21% of
inosine-treated cells with axons <5 cell diameters in length.
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Fig. 5.
Purines stimulate expression of the
growth-associated protein GAP-43. a-d, a polyclonal
rabbit antibody generated to recombinant zebrafish GAP-43 was used with
a fluorescein-conjugated secondary antibody to visualize GAP-43 in
goldfish RGCs treated for 6 days with defined medium alone
(a), AF-1 (b), inosine (100 µM,
c), or guanosine (100 µM, d).
e-g, same fields as in a-c stained with DAPI to
visualize cell nuclei. h, Western blot of proteins from
approximately 106 RGCs treated with medium alone (L-15),
AF-1, or inosine (100 µM) and probed with the anti-GAP-43
antibody. An immunoreactive band at Mr = 43 kDa,
the expected migration position of GAP-43, increases in response to
AF-1 or inosine (iso).
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Fig. 6.
Inosine and AF-1 both stimulate growth
through a purine-sensitive mechanism. a, at 10 µM, the purine analog 6-TG blocks growth induced by AF-1
(lane 2 versus lane 1; p < 0.001), reduces the effects of 25 µM inosine by 50%
(lane 4 versus lane 3), and has no effect on
growth stimulated by 100 µM inosine (lane 5 versus
lane 6) or guanosine (lane 8 versus lane 7).
Inosine (100 µM) restored growth induced by AF-1 in the
presence of 10 µM 6-TG (lane 10) back to its
original level, which is significantly higher than that induced by 100 µM inosine alone (**, p < 0.01).
b, 6-TG had no effects on cell survival. c, AF-1
and inosine have partially additive effects. In a 2 × 3 design,
outgrowth was assessed for AF-1 and inosine, each at 0, its
EC50, or saturating concentrations. Although the effects of
half-maximal concentrations of each were additive (lane 5),
growth reached a plateau in the presence of higher concentrations of
each (lanes 6, 8, and 9).
d, inhibition of AF-1-stimulated growth by 6-TG (10 µM) is not restored by the purine transport blockers NBTI
(N, 20 µM) or dipyridamole (dipyr,
D, 10 µM). The inhibitory effects of 6-TG are
not mimicked by the free radical scavengers, -tocopherol
(-toc, -t, 30 µM) or
glutathione -methyl ester (MEG, 100 µM).
Ino-0, -10, -25, -50, and
-100, 0, 10, 25, 50, and 100 µM inosine;
Guo-100, 100 µM guanosine.
-tocopherol (30 µM),
and glutathione -methyl ester (100 µM). Unlike 6-TG,
these agents had no effect on outgrowth stimulated by AF-1 (Fig.
6d). Another question was whether inosine might block the
inhibitory effect of 6-TG on outgrowth by interfering with its
transport into cells. This was investigated by examining whether the
two transport inhibitors that block the positive effects of inosine would also block the negative effects of 6-TG. Neither NBTI (20 µM) nor dipyridamole (10 µM) prevented 6-TG
from blocking outgrowth stimulated by AF-1 (Fig. 6d).
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Fig. 7.
Effects of purines on rat retinal ganglion
cells. RGCs from postnatal day 8 rats were isolated by
immunopanning (15) and treated for 2 days with defined medium (5)
containing 5 µM forskolin and 26 mM
bicarbonate (a) or the same media plus 25 µM
inosine (b).Scale bar equals 50 microns. c, overall,
inosine increases growth above base line by 52% (lane 2 versus
lane 1; **, p < 0.01); CNTF has a more pronounced
effect (lane 3; ***, p < 0.001) that is
blocked by 6-TG (lane 5) but largely restored upon the
addition of inosine (*, p < 0.05). d, cell
survival is unaffected by inosine (lane 2) but is increased
by CNTF (lanes 3-6) irrespective of whether 6-TG is absent
(lanes 3 and 4) or present (lanes 5 and 6).
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Fig. 8.
Preliminary studies on signal transduction
pathways activated by inosine and AF-1. Top, outgrowth
induced by inosine (100 µM) was inhibited by about 50%
by the MEK-1/-2 inhibitor PD 098059 (50 µM) or by the
inhibitor of PI 3-kinase, LY 294002 (20 µM); when the two
were combined, outgrowth was blocked completely. In contrast, neither
affected growth stimulated by AF-1. However, both inhibitors combined
decreased the effects of AF-1 by 40%. Bottom, cell survival
was not significantly affected by the inhibitors. ***, differences
significant at p < 0.002.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-1 tubulin3 and
up-regulated expression of the cell adhesion molecule
L1,4 both of which are
activated by regeneration in vivo.
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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